Proc. Natl. Acad. Sci. USA Vol. 76, No. 5, pp. 2185-2189, May 1979
Biochemistry
Activation of phenotypic expression of human globin genes from nonerythroid cells by chromosome-dependent transfer to tetraploid mouse erythroleukemia cells (cDNA/RNA/gene regulation/gene transfer/cell fusion)
ALBERT DEISSEROTH AND DON HENDRICK Experimental Hematology Section, Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205
Communicated by Marshall Warren Nirenberg, February 14, 1979
ABSTRACT Chromosome-dependent gene transfer mediated by cell fusion was used to show that it is possible to activate phenotypic expression of human a globin genes derived from nonerythroid cells. Hybrid cells containing the human a globin structural genes were derived by fusion of populations of adult human peripheral blood mononuclear cells (devoid of identifiable erythroid cells) with adenine phosphoribosyltransferase-deficient mouse erythroleukemia cells that contained close to a tetraploid complement of mouse chromosomes. The hybrid cells retained a near tetraploid complement of mouse chromosomes but had lost 80% of the chromosomes of the human parent cell. All of these hybrid cells and their subclones, which contained human chromosome 16, exhibited synthesis of human a globin chains. Human a globin mRNA was also demonstrated to be present in one of these hybrid cells by RNA'cDNA molecular hybridization analysis. We conclude that the mechanism responsible for restricting expression of the human globin genes in nonerythroid cells is not irreversible, at least for those globin structural genes that are actively transcribed in erythroid cells during adult life. Moreover, some genetic factor or process in the tetraploid mouse erythroleukemia cell is, under the conditions of our experiments, capable of reactivating phenotypic expression (production of globin chains) of human globin genes derived from nonerythroid hematopoietic cells after chromosome-dependent gene transfer. Although the chromosomal locations of the human globin genes have been determined (1-3), the genetic mechanisms responsible for the maintenance of balanced synthesis of a and non-a globin chains in erythroid cells and for the differential expression of the human : and y non-a chains during fetal and adult life are at present unknown. The process that provides for the expression of globin genes in erythroid cells and restricts the expression of these genes in nonerythroid cells also remains to be elucidated. In order to develop a system in which such questions could be studied, our laboratory has devised a chromosome-dependent gene transfer system which is mediated by cell fusion. This system provides for the permanent retention in mouse erythroleukemia cells of human chromosomes bearing the human a globin structural genes (4, 5). Data obtained by using this system has shown that hybrid mouse erythroleukemia cells that contain human chromosome 16 exhibit persistent expression of human a globin chains when the population of human donor cells is dominated by erythroid cells (4-6). Using differentiated markers other than hemoglobin, other workers have shown that hybrid cells that retained equal numbers of chromosomes from differentiated and undifferentiated parent cells did not contain the marker of cell differentiation originally present in the differentiated parent cell (7). Our own work has shown that hybrid cells derived by fusion of diploid mouse erythroleukemia cells with human fibroblasts, which retained a diploid complement
of mouse erythroleukemia chromosomes and a hyperdiploid complement of human chromosomes, did not exhibit production of mouse or human globin mRNA even though the globin structural genes of both species were present (8, 9). Other workers have produced similar findings by using hybrid cells derived by fusion of diploid mouse erythroleukemia cells and mouse fibroblasts (10, 11). In order to determine whether there are factors or genetic mechanisms in the mouse erythroleukemia cell that can, under certain conditions, result in the activation of expression of human globin genes derived from nonerythroid cells, we have now isolated hybrid cells derived by fusion of tetraploid mouse erythroleukemia cells with human peripheral blood mononuclear cells. These hybrid cells contain a near tetraploid complement of mouse chromosomes but have retained only a few human chromosomes among which is that bearing the human a globin structural gene. The design of this experiment was based on earlier observations by other workers (7, 12) in which fusion of a tetraploid differentiated cell and a diploid undifferentiated cell resulted in persistence of expression of markers (dopamine oxidase, albumin) originally present in the differentiated parent as well as activation of expression of the corresponding genes from the undifferentiated parent cell (12). The hybrid cells we have isolated contain human a globin chains and thus exhibit expression through the level of translation of human a globin genes that were derived from nonerythroid hematopoietic cells. These experiments indicate, therefore, that the restriction of expression of globin genes in nonerythroid cells can be fully reversed by introduction of these structural genes into tetraploid mouse erythroleukemia cells. MATERIALS AND METHODS Isolation of Hybrid Cells. A mouse erythroleukemia cell that contained an average of 72 mouse chromosomes (referred to as a "near tetraploid" complement of mouse chromosomes) was isolated by fusion of Friend mouse erythroleukemia clone 745, which is resistant to 6-thioguanine, with Friend mouse erythroleukemia clone 707, which is resistant to bromodeoxyuridine, as described (8). Cloned populations of this hybrid cell were then exposed to increasing concentrations of diaminopurine until resistance to 100 /ig of this agent per ml was observed (4). The diaminopurine-resistant cells were then exposed to increasing concentrations of 2-fluoroadenine until resistance to 100 tug of 2-fluoroadenine per ml was established (4). Populations of these cells, after cloning in 100 ,g of 2-fluoroadenine per ml, exhibited less than 0.03% of the activity of the enzyme adenine phosphoribosyltransferase (APRT, EC 2.4.2.7), which is found in mouse erythroleukemia cells before selection, when assayed as described (4, 13).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: APRT, adenine phosphoribosyltransferase; Crot; initial concentration of DNA (moles of nucleotide per liter) X time (seconds); Crot1/2, Crot necessary for 50% hybridization; Me2SO, dimethyl sulf-
oxide.
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Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Deisseroth and Hendrick
Mononuclear cells were isolated from the peripheral blood of normal individuals by Ficoll-Hypaque centrifugation as described by Boyum (14). A mixed population of 2 X 107 human peripheral blood mononuclear cells and 5 X 106 APRT-deficient mouse erythroleukemia cells, containing a near tetraploid complement of mouse chromosomes, was rinsed twice in Dulbecco's modification of Eagle's medium without fetal calf serum. After centrifugation and careful removal of all supernatant fluid, 0.7 ml of the medium, which was 40% in polyethylene glycol (molecular weight range 950-1050) and 10% in dimethyl sulfoxide (Me2SO), was added over 10 sec with gentle stirring of the cells, following a modification of the procedure of Davidson et al. (15). After incubation for 30 sec at room temperature, 1 ml of medium was added over 1 min with gentle stirring. Additional (4 ml) medium was added over the succeeding 2 min. Then, 15 ml of medium was added at once and the suspension was centrifuged. The pellet was gently resuspended in medium supplemented with 10% fetal calf serum, and the suspension was incubated for 24 hr at 370C. After centrifugation and removal of the supernatant, the cells were then diluted into selective medium containing alanosine and adenine as the only purine source (4) and plated in 20 Falcon T 75 flasks. Hybrid populations that contained human chromosome 16 were then cloned in suspension.
Chromosomal Analysis. Three to four weeks following fusion, populations of cells were screened fot the presence of intact human chromosomes by Giemsa/trypsin banding-Hoechst 33258 sequential staining, as described (2, 3, 4, 16, 17), or by a second method in which simple Giemsa staining replaced the exposure to Giemsa/trypsin (4). CM-Cellulose Column Chromatography of Globin Chains. Hybrid populations (5 X 106 cells) incubated for 5 days in 1.25% Me2SO were exposed for 6 hr to 0.5 mCi (1 Ci = 3.7 X 1010 becquerels) of [3H]leucine (specific activity, 50 mCi/mmol) in 25 ml of medium. Fractionation of hemoglobin from other radioactively labeled cytoplasmic proteins and chromatography of the globin chains after heme extraction was carried out as described (4, 6). APRT Analysis. Cell pellets from stationary phase cultures were processed and analyzed for the presence of human and mouse ARPT as described by Tischfield and Ruddle (13). Analysis of Cytoplasmic RNA for Human Globin Coding Sequences. Cytoplasmic extracts were obtained from cultured cells as described (4). The RNA was purified by the method of Aviv and Leder (18). Human a globin mRNA, cytoplasmic poly(A)-containing RNA, and cDNA were prepared as described (4, 19). Human a globin cDNA was further purified by preparative hybridization and hydroxylapatite chromatography. First, an analytical scale Crot [initial concentration of RNA (moles/liter) X time (seconds)] analysis was performed by annealing a chain-enriched cDNA (80% pure) to a 4-fold excess of globin mRNA purified from reticulocytes of a donor with hemoglobin H disease (2). The resulting Crot curve showed transitions at CrOtj/2 (CrOt necessary for 50% hybridization) = 4.5 X 10-2 and 2.5 X 10-1 M X sec. The preparative reaction was performed with 3.6 ,ug of hemoglobin H mRNA (,B to a globin mRNA ratio = 4:1) and 20 ng of the a chain-enriched cDNA incubated in 750 ul of 50% formamide buffer (see below). This mixture was taken to a Crot value of 6 X 10-2 at 62°C, diluted with 7.5 ml of 50 mM sodium phosphate, pH 6.8/0.4% sodium dodecyl sulfate, and applied to a 0.7-ml bed volume hydroxylapatite column with 140 mM sodium phosphate, pH 6.8/0.4% sodium dodecyl sulfate. The hybrid was eluted with 480 mM sodium phosphate, pH 6.8/0.4% sodium dodecyl sulfate. cDNA was recovered as described (2). This method of purifying the a globin cDNA freed it of essentially
all cross-hybridizing human f3 globin coding sequences. The final product, [3H]cDNA, migrated as a single peak of radioactivity on 5% polyacrylamide gels in 99% formamide and had a molecular weight of 230,000 and a specific activity of 4.27 X 104 dpm/ng (1 dpm = 16.7 mBq). In the nucleic acid molecular hybridization assays, there were 2-jul aliquots of cDNA (about 0.1 ng) in 0.5 M NaCl/25 mM Hepes/0.5 mM EDTA/ 50% formamide (pH 6.8), which was used to dissolve appropriate amounts of salt-free, freeze-dried RNA. The mixtures were sealed in siliconized capillary tubes, denatured for 10 min at 950C, and then allowed to hybridize for 90 hr at 660C. Hybridization was terminated abruptly by plunging the capillaries into a dry ice/acetone bath. Hybrids were assayed as described (20) by their resistance to S1 nuclease. RESULTS
Chromosomal composition of hybrid cells derived by fusion of tetraploid mouse erythroleukemia cells and human peripheral blood leukocytes Previous work in our laboratory had shown that isolation of hybrid cells derived by fusion of mouse erythroleukemia cells deficient in APRT with human cells in selective medium supplemented with alanosine and adenine results in hybrid populations in which human chromosome 16 is present at a frequency of 80% (4). In the present study, mononuclear cells isolated from the peripheral blood of normal adult individuals by Ficoll-Hypaque centrifugation (14) were fused in the presence of polyethylene glycol and Me2SO with APRT-deficient mouse erythroleukemia cells that contained a near tetraploid complement of mouse chromosomes, as shown in Table 1. Analysis of metaphase spreads of the two hybrid populations resulting from this fusion by the species-specific stains showed that the hybrids had retained a tetraploid complement of mouse chromosomes and less than 15% of the chromosomes present in the original human parent cells. An example of a single metaphase spread of hybrid population 1, which was stained by two different species-specific stains, is shown in Fig. 1. As
..,
16
*
.'
A~6
ve
eA
'
FIG. 1. Single metaphase spread of hybrid population 1. (Left) Exposed to Hoechst 33258, which stains the human chromosomes with a uniform intensity but stains mouse chromosomes more intensely near the centromeres and less intensely along the arms. (Right) The same metaphase spread was stained with Giemsa after exposure to trypsin, which generates banding specific for each human and mouse chromosome. Human chromosome 16 is indicated. A few human chromosomes in addition to human chromosome 16 that were found in this metaphase spread are identified by additional small black arrows.
Biochemistry:
Deisseroth and Hendrick
Proc. Natl. Acad. Sci. USA 76 (1979)
2187
Table 1. Properties of hybrid cells derived by fusion of near tetraploid mouse erythroleukemia cells with human peripheral leukocytes
Total mouse chromosomes per cell*
% cells containing human chromosome 2 Cell line 3 4 6 9 11 8 12 21 10 13 16 20 Unfused parent mouse erythro72 leukemia cell 0 0 0 0 0 0 0 0 0 0 0 0 0 Hybrid cell 1 81 10 45 5 (uncloned) 60 25 15 65 15 45 5 85 35 0 Hybrid cell 2 71 4 4 (uncloned) 33 0 0 0 0 0 0 4 90 0 19 * Thirty metaphase spreads of each uncloned hybrid population were analyzed for the composition of human chromosomes. Human chromosomes that are not listed were not observed in any of the metaphase spreads examined.
shown by the data in Table 1 and Fig. 1, nearly every metaphase spread analyzed contained human chromosome 16 (which bears the human a globin structural gene) in spite of the extensive loss of human chromosomes. The frequency of the other human chromosomes retained by these hybrid cells is also presented in Table 1. Independent confirmation of the presence of human chromosome 16 in these hybrid populations was established by electrophoresis of protein extracts of the hybrid cells and autoradiographic analysis of the electrophoretograms for APRT. This enzyme is normally constitutively synthesized and is known to be coded for by human chromosome 16 (13). As shown by the data presented in Table 1 and Fig. 2, hybrid cell populations 1 and 2, which contain human chromosome 16, also contained the human form of APRT. Analysis of cytoplasmic RNA of hybrid cells for the
presence of human a globin mRNA
Hybrid populations 1 and 2, parent mouse erythroleukemia cells, and a continuously proliferating human lymphocytic cell line, Molt-4 (21), were incubated in 1.25% Me2SO for 5 days. This treatment is known to result in induction of expression of the mouse globin genes in mouse erythroleukemia cells (22, 23). Cytoplasmic poly(A)-containing RNA was purified from the Me2SO-induced hybrid cells and incubated with highly purified human a globin cDNA. Hybridization between mouse a globin mRNA and human a globin cDNA does not occur under the conditions used in our experiments, as reported by us (4) and shown in Fig. 3. The data presented in Fig. 3 also show that the cytoplasmic poly(A)-containing RNA of hybrid population 1 contained human a globin coding sequences after induction Origins
1
2
3
4
5
80 70 °- 60 *550
FIG. 2. Autoradiograph of a polyacrylamide slab gel for detection of human and mouse APRT in hybrid cell extracts. Extracts of mouse fibroblasts (lane 1), APRT-deficient mouse erythroleukemia cells (lane 3), hybrid cell 1 (lane 4), and human lymphocytes (lane 6) were subjected to electrophoresis and then exposed to the APRT reaction mixture. Lane 2 contained bromophenol blue and all other lanes (except for lane 5) contained approximately 10 ,g of protein.
0 40
+
28
+
0
1
C
C
0
40t
.C30 R 20 10 1 2 3 4 5 6*
300 0
Human APRT
in Me2SO. One can conclude from the ratio of the two abscissae (a to 0) in Fig. 3 that human a globin mRNA represented about 1 in 300 cytoplasmic poly(A)-containing RNA sequences in the hybrid cells. Also presented in Fig. 3 are control hybridization assays which included the incubation of human a globin cDNA under the same conditions with cytoplasmic poly(A)-containing RNA from the Me2SO-induced parent tetraploid mouse erythroleukemia cells and from Molt-4 after incubation with Me2SO. There was no cross hybridization of mouse erythroleukemia poly(A)-containing RNA with human (v globin cDNA under the conditions employed for the experiments. From our control assays, we know that the poly(A)containing RNA from the Me2SO-induced mouse parent cells contains about 1% mouse globin mRNA sequences. As shown in Fig. 3, there were no detectable human a globin coding sequences in the poly(A)-containing RNA from the human lymphocytic cell line Molt-4 at RNA to cDNA ratios up to 5000. When poly(A)-containing RNA from Molt-4 cells was used to extend the RNA to cDNA ratios up to 1.5 X 105, we still were unable to detect any human (v globin coding sequences in the RNA from these Me2SO-induced human lymphocytic cells
6
Q Co
22
900 1500
0
2500 5000 AD RNA/cDNA FIG. 3. Hybridization of human a globin cDNA to cytoplasmic RNA extracted from Me2SO-induced hybrid cell 1, which contained a near tetraploid complement of mouse chromosomes and human chromosome 16. Each 2-Ml incubation mixture contained 0.1 ng of human a globin cDNA and was incubated for 90 hr at 660C. The ratio of RNA to cDNA in each incubation is plotted versus the percentage of cDNA that formed a hybrid with RNA. Hybrid formation was assayed by resistance to S1 nuclease. In addition to human a globin cDNA, the incubation mixture contained: 0, human a globin mRNA; 0, cytoplasmic poly(A)-containing RNA from hybrid cells; 03, cytoplasmic poly(A)-containing RNA from parent mouse erythroleukemia cells; A, cytoplasmic poly(A)-containing RNA from Molt-4, a human
lymphocyte cell line.
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Proc. Natl. Acad. Sci. USA 76 (1979)
(unpublished results). Thus, transfer of the human a globin from nonerythroid donor cells, which were devoid of human globin coding sequences in cytoplasmic RNA, to tetraploid mouse erythroleukemia cells resulted in the activation of human globin gene expression. Study of human a globin chain synthesis in hybrid
genes
a
a
cells
The two hybrid tetraploid mouse erythroleukemia cell populations that contained human chromosome 16 were analyzed for the presence of human a globin chains by exposure of the Me2SO-induced hybrid cells to [3H]leucine for 6 hr, as described in Methods and by us previously (4-6). After purification of labeled hemoglobin from the hybrid cells and extraction of heme, the resulting globin chains were fractionated on columns of CM-cellulose. As shown by the data presented in Fig. 4 and Table 2, both of these hybrid populations exhibited expression of human a globin chains as well as mouse a and # globin chains.
We then analyzed four subclones isolated from hybrid populations 1 and 2, which had been derived from four independent fusion events and which contained human chromosome 16 in 100% of the cells analyzed (see Table 2). In all four subclones, induction by Me2SO resulted in the appearence of human a globin chains in addition to mouse a and globin chains. Thus, the hybrid cells and their subclones, which retained a tetraploid complement of mouse erythroleukemia chromosomes as well as human chromosome 16, reproducibly exhibit phenotypic expression, through the level of globin chain synthesis, of the human a globin genes derived from nonerythroid cells.
DISCUSSION The experiments summarized in this report have documented that the mechanisms responsible for the restriction of expression of human globin genes in nonerythroid cells are reversible by transfer of these genes into a mouse erythroleukemia cell in which a near tetraploid complement of mouse chromosomes has been retained. In addition, the correlation in the cell hybrids reported in this study between the production of human globin chains and the presence of human chromosome 16 confirms recent publications from our laboratory (2, 4) in which a
0
50
100 Fraction
150
FIG. 4. CM-cellulose chromatography of [3H]leucine-labeled globin chains in cell hybrid 1. This hybrid cell was derived by fusion of APRT-deficient mouse erythroleukemia cells with human peripheral blood leukocytes in which retention of a near tetraploid complement of mouse chromosomes as well as human chromosome 16 had occurred. A 6-hr labeling period was conducted after 5 days of incubation in Me2SO.
Table 2. Content of human a globin genes and human chromosome 16 in subclones of hybrid mouse erythroleukemia cells Cells positive Human a globin for human Hybrid chromosome 16* chains
Hybrid 1: Uncloned Clone 9 Clone 10
85 100 100
+ + +
Hybrid 2: Uncloned Clone 1 Clone 3
90 100 100
+ + +
* Twenty metaphase spreads of each hybrid clone and 30 metaphase spreads of each uncloned hybrid population were analyzed for human chromosome content.
the localization of the human a structural genes to this chromosome was established by independent means. The population used as the donor for the human globin structural genes was obtained from human peripheral blood by Ficoll-Hypaque centrifugation. None of the 50,000 cells studied was positive when stained for hemoglobin with benzidine, and the frequency of committed erythroid precursor cells cannot be placed above 1:5000 (24). Thus, the probability that the donor cells for the four independent fusion events derived from hybrid populations 1 and 2 were all of erythroid origin is less than 1 X 10-14. Moreover, measurements by other workers have shown that populations of peripheral blood leukocytes drawn from normal individuals in a manner similar to that used in our studies do not contain detectable levels of human globin mRNA (25). Studies by Miller et al. (26) and studies in our own laboratory (see Fig. 3) have shown that established cell lines exhibiting markers of lymphoid or myeloid (unpublished results) origin do not contain globin coding sequences in cytoplasmic RNA even after incubation in Me2SO. All of these studies taken together have established that the human globin genes transferred to mouse erythroleukemia cells in this report were derived from donor cells in which there were no detectable levels of human globin mRNA or globin chains. Miller et al. (26) have shown that lymphoblast cell lines in which there are no detectable levels of human globin mRNA do share a property in common with erythroid cells-that of complete sensitivity of the globin genes to digestion with DNase 1. Thus, the genetic mechanisms governing restriction of phenotypic expression of globin genes in nonerythroid hematopoietic cells must not rest on the configuration of the globin genes in so far as it is measurable by DNase 1 sensitivity. As discussed above, previous work with cell hybrids suggested that fusion of a diploid differentiated cell with a diploid undifferentiated cell results in hybrid cells in which loss of phenotypic expression is observed (7-9). Experiments by Axelrod et al. (27) showed that maintenance of the phenotype of hemoglobin inducibility by Me2SO is observed in hybrid cells derived by fusion of near tetraploid mouse erythroleukemia cells with human fibroblasts. Hybrid cells isolated by the same group of workers (derived by fusion of human fibroblasts with mouse erythroleukemia cells), which retained a near tetraploid complement of mouse chromosomes as well as human chromosome 11, contained human ( globin coding sequences in the cytoplasmic RNA after induction by Me2SO (28). Even though the 3 and Ty globin structural genes have been shown to be closely linked on chromosome 11 (3, 29), these hybrid cells contained no detectable levels of human y globin coding sequences in their cytoplasmic RNA (28). No data was presented
Biochemistry:
Deisseroth and Hendrick
by these authors however to establish the presence or absence of human d globin chains (28). Thus, one is unablf to conclude from the work of these authors whether they had isolated hybrid cells in which activation of full phenotypic expression of human /3 globin genes from nonerythroid cells had occurred. It is not known at this time whether the different patterns of differentiated gene expression seen in the fusion experiments summarized in this discussion arise primarily from the ploidy of the recipient mouse erythroleukemia cell or depend upon the retention or loss of chromosomes of human origin, which may exert a negative regulatory effect on the expression of globin genes in nonerythroid cells. Our experiments as well as those cited above (28) do suggest however that the mechanisms that mediate the differential expression of the two non-a (/3 and y) globin structural genes in adult and fetal life depend on a genetic process that is different from that responsible for the restriction of expression of globin genes in nonerythroid cells. This appears to be the case, because transfer of human chromosomes 11 and 16 from nonerythroid human donor cells to tetraploid mouse erythroleukemia cells resulted in activation of expression of a and / but not Sy globin structural genes. The experiments of Young et al. (30), which showed that both the y and : globin structural genes are equally sensitive to the digestive action of DNase 1 in adult erythroid cells, were interpreted by the authors as consistent with regulation of expression of y and /3 globin genes at the transcriptional or posttranscriptional level. Wood et al. (31) have also presented evidence that posttranscriptional events are responsible for the differential expression at the phenotypic level of the human /3 and 6 globin genes in adult erythroid cells. The presence of a globin chains in the hybrid tetraploid mouse erythroleukemia cells we have isolated, which also contain human a globin genes derived from nonerythroid cells, decisively demonstrates that the process that mediates restriction of expression of human globin genes in nonerythroid cells is not irreversible. These data also establish that transfer of the chromosomes bearing the human globin genes from nonerythroid cells to the near tetraploid mouse erythroleukemia cells results in activation of phenotypic expression of these genes, as shown by the synthesis of human globin chains. Application of the technology of restriction endonuclease mapping and plasmid cloning (32-34) to the study of these populations of hybrid cells may contribute structural information that would help clarify the precise mechanisms responsible for these findings. Further study of the experimental activation of phenotypic expression of human globin genes derived from nonerythroid cells may also result in information that will be of use in developing treatments for disease states in man in which activation of nonexpressed globin genes in erythroid cells would be of potential benefit. The authors recognize the expert technical assistance of Mary Blenkush and Sylvon Von Der Pool. Avian myeloblastosis virus RNA-directed DNA polymerase was obtained through Program Resources and Logistics, Viral Oncology Program, National Cancer Institute. 1.
Deisseroth, A., Velez, R. & Nienhuis, A. (1976) Science 191,
1262-1263. 2. Deisseroth, A., Nienhuis, A., Turner, P., Velez, R., Anderson, W. F., Ruddle, F., Lawrence, J., Creagen, R. & Kucherlapati, R. (1977) Cell 12,205-218.
Proc. Natl. Acad. Sci. USA 76 (1979)
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3. Deisseroth, A., Nienhuis, A., Lawrence, J., Giles, R., Turner, P. & Ruddle, F. (1978) Proc. Natl. Acad. Sci. USA 75, 14561460. 4. Deisseroth, A. & Hendrick, D. (1978) Cell 15,55-63. 5. Deisseroth, A. & Hendrick, D. (1978) in Cellular and Molecular Regulation of Hemoglobin Switching, eds. Stamatoyannopoulos, G. & Nienhuis, A. (Grune & Stratton, New York), pp. 761777. 6. Deisseroth, A., Barker, J., Anderson, W. F. & Nienhuis, A. (1975) Proc. Natl. Acad. Sci. USA 72,2682-2686. 7. Davidson, R. L. (1974) in Somatic Cell Hybridization, eds. Davidson, R. L. & De La Cruz, F. (Raven, New York), pp. 137-150. 8. Deisseroth, A., Burk, R., Picciano, D., Minna, J., Anderson, W. F. & Nienhuis, A. (1975) Proc. Natl. Acad. Sci. USA 72, 11021106. 9. Deisseroth, A., Velez, R., Burk, R., Minna, J., Anderson, W. F. & Nienhuis, A. (1976) Somatic Cell Genet. 2,373-384. 10. Orkin, S., Harosi, R. & Leder, P. (1975) Proc. Natl. Acad. Sci. USA 72, 98-102. 11. Conscience, J. F., Ruddle, F., Skoultchi, A. & Darlington, G. (1977) Somatic Cell Genet. 3, 157-172. 12. Darlington, G., Bernhard, H. & Ruddle, F. (1974) Science 185, 859-862. 13. Tischfield, J. & Ruddle, F. (1974) Proc. Natl. Acad. Sci. USA 71, 45-49. 14. Boyum, A. (1968) Scand. J. Clin. Lab. Invest. 21, Suppl. 97, 77-89. 15. Davidson, R. L., O'Malley, K. & Wheeler, T. B. (1976) Somatic Cell Genet. 2, 271-280. 16. Kozak, C., Lawrence, J. & Ruddle, F. (1977) Exp. Cell Res. 105, 109-117.
17. Yoshida, M. C., Ikeuchi, R. & Sasake, M. (1975) Proc. Jpn. Acad. 51, 184-187. 18. Aviv, J. & Leder, P. (1972) Proc. Natl. Acad. Sci. USA 69, 1408-1412. 19. Forget, B. G., Housman, D., Benz, E. J. & MaCaffrey, R. P. (1975) Proc. Natl. Acad. Sci. USA 72,984-988. 20. Jackson, J., Tolstoshev, P., Williamson, R. & Hendrick, D. (1976) Nucleic Acids Res. 3, 2019-2026. 21. Minowada, J., Ohnuma, T. & Moore, G. (1972) J. Natl. Cancer Inst. 49, 891-894. 22. Friend, C., Scher, W., Holland, J. C. & Sato, T. (1971) Proc. Natl. Acad. Sci. USA 68,378-382. 23. Cooper, M. C., Levy, J., CantorJ. N., Marks, P. A. & Rifkind, R. A. (1974) Proc. Natl. Acad. Sci. USA 71,1677-1680. 24. Clarke, B. J. & Housman, D. (1977) Proc. Natl. Acad. Sci. USA 74, 1105-1109. 25. Gianni, A. M., Dalla, F. R., Polli, E., Merisio, I., Giglioni, B., Comi, P. & Ottolenghi, S. (1978) Nature (London) 274, 610-611. 26. Miller, D. N., Turner, P., Nienhuis, A., Axelrod, D. C. & Gopalakrishnan, T. V. (1978) Cell 15,511-521. 27. Axelrod, D. C., Gopalakrishnan, T. V., Willing, M. & Anderson, W. F. (1978) Somatic Cell Genet. 4, 157-168. 28. Anderson, W. F., Willing, M. D. & Nienhuis, A. (1978) Blood 52, Suppl. 1, p. 108. 29. Smith, D. H., Clegg, J. B., Weatherall, D. J. & Gilles, H. M. (1973) Nature (London) New Biol. 246, 184-188. 30. Young, N., Benz, E. J., Jr., Kantor, J. A., Kretschmer, P. & Nienhuis, A. (1978) Proc. Natl. Acad. Sci. USA 75, 58845888. 31. Wood, W. G., Old, J. M., Roberts, A. V. S., Clegg, J. B. & Weatherall, D. J. (1978) Cell 15, 437-446. 32. Mears, J. G., Ramirez, F., Leibowitz, D. & Bank, A. (1978) Cell 15, 15-23. 33. Embury, S. H., Dozy, A. M. M. & Kan, Y. W. (1978) Blood 52, Suppl. 1, p. 111. 34. Mulligan, R. C., Howard, B. H. & Berg, P. (1979) Nature (London) 277, 108-114.
Biochemistry
Activation of phenotypic expression of human globin genes from nonerythroid cells by chromosome-dependent transfer to tetraploid mouse erythroleukemia cells (cDNA/RNA/gene regulation/gene transfer/cell fusion)
ALBERT DEISSEROTH AND DON HENDRICK Experimental Hematology Section, Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205
Communicated by Marshall Warren Nirenberg, February 14, 1979
ABSTRACT Chromosome-dependent gene transfer mediated by cell fusion was used to show that it is possible to activate phenotypic expression of human a globin genes derived from nonerythroid cells. Hybrid cells containing the human a globin structural genes were derived by fusion of populations of adult human peripheral blood mononuclear cells (devoid of identifiable erythroid cells) with adenine phosphoribosyltransferase-deficient mouse erythroleukemia cells that contained close to a tetraploid complement of mouse chromosomes. The hybrid cells retained a near tetraploid complement of mouse chromosomes but had lost 80% of the chromosomes of the human parent cell. All of these hybrid cells and their subclones, which contained human chromosome 16, exhibited synthesis of human a globin chains. Human a globin mRNA was also demonstrated to be present in one of these hybrid cells by RNA'cDNA molecular hybridization analysis. We conclude that the mechanism responsible for restricting expression of the human globin genes in nonerythroid cells is not irreversible, at least for those globin structural genes that are actively transcribed in erythroid cells during adult life. Moreover, some genetic factor or process in the tetraploid mouse erythroleukemia cell is, under the conditions of our experiments, capable of reactivating phenotypic expression (production of globin chains) of human globin genes derived from nonerythroid hematopoietic cells after chromosome-dependent gene transfer. Although the chromosomal locations of the human globin genes have been determined (1-3), the genetic mechanisms responsible for the maintenance of balanced synthesis of a and non-a globin chains in erythroid cells and for the differential expression of the human : and y non-a chains during fetal and adult life are at present unknown. The process that provides for the expression of globin genes in erythroid cells and restricts the expression of these genes in nonerythroid cells also remains to be elucidated. In order to develop a system in which such questions could be studied, our laboratory has devised a chromosome-dependent gene transfer system which is mediated by cell fusion. This system provides for the permanent retention in mouse erythroleukemia cells of human chromosomes bearing the human a globin structural genes (4, 5). Data obtained by using this system has shown that hybrid mouse erythroleukemia cells that contain human chromosome 16 exhibit persistent expression of human a globin chains when the population of human donor cells is dominated by erythroid cells (4-6). Using differentiated markers other than hemoglobin, other workers have shown that hybrid cells that retained equal numbers of chromosomes from differentiated and undifferentiated parent cells did not contain the marker of cell differentiation originally present in the differentiated parent cell (7). Our own work has shown that hybrid cells derived by fusion of diploid mouse erythroleukemia cells with human fibroblasts, which retained a diploid complement
of mouse erythroleukemia chromosomes and a hyperdiploid complement of human chromosomes, did not exhibit production of mouse or human globin mRNA even though the globin structural genes of both species were present (8, 9). Other workers have produced similar findings by using hybrid cells derived by fusion of diploid mouse erythroleukemia cells and mouse fibroblasts (10, 11). In order to determine whether there are factors or genetic mechanisms in the mouse erythroleukemia cell that can, under certain conditions, result in the activation of expression of human globin genes derived from nonerythroid cells, we have now isolated hybrid cells derived by fusion of tetraploid mouse erythroleukemia cells with human peripheral blood mononuclear cells. These hybrid cells contain a near tetraploid complement of mouse chromosomes but have retained only a few human chromosomes among which is that bearing the human a globin structural gene. The design of this experiment was based on earlier observations by other workers (7, 12) in which fusion of a tetraploid differentiated cell and a diploid undifferentiated cell resulted in persistence of expression of markers (dopamine oxidase, albumin) originally present in the differentiated parent as well as activation of expression of the corresponding genes from the undifferentiated parent cell (12). The hybrid cells we have isolated contain human a globin chains and thus exhibit expression through the level of translation of human a globin genes that were derived from nonerythroid hematopoietic cells. These experiments indicate, therefore, that the restriction of expression of globin genes in nonerythroid cells can be fully reversed by introduction of these structural genes into tetraploid mouse erythroleukemia cells. MATERIALS AND METHODS Isolation of Hybrid Cells. A mouse erythroleukemia cell that contained an average of 72 mouse chromosomes (referred to as a "near tetraploid" complement of mouse chromosomes) was isolated by fusion of Friend mouse erythroleukemia clone 745, which is resistant to 6-thioguanine, with Friend mouse erythroleukemia clone 707, which is resistant to bromodeoxyuridine, as described (8). Cloned populations of this hybrid cell were then exposed to increasing concentrations of diaminopurine until resistance to 100 /ig of this agent per ml was observed (4). The diaminopurine-resistant cells were then exposed to increasing concentrations of 2-fluoroadenine until resistance to 100 tug of 2-fluoroadenine per ml was established (4). Populations of these cells, after cloning in 100 ,g of 2-fluoroadenine per ml, exhibited less than 0.03% of the activity of the enzyme adenine phosphoribosyltransferase (APRT, EC 2.4.2.7), which is found in mouse erythroleukemia cells before selection, when assayed as described (4, 13).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: APRT, adenine phosphoribosyltransferase; Crot; initial concentration of DNA (moles of nucleotide per liter) X time (seconds); Crot1/2, Crot necessary for 50% hybridization; Me2SO, dimethyl sulf-
oxide.
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Biochemistry: Deisseroth and Hendrick
Mononuclear cells were isolated from the peripheral blood of normal individuals by Ficoll-Hypaque centrifugation as described by Boyum (14). A mixed population of 2 X 107 human peripheral blood mononuclear cells and 5 X 106 APRT-deficient mouse erythroleukemia cells, containing a near tetraploid complement of mouse chromosomes, was rinsed twice in Dulbecco's modification of Eagle's medium without fetal calf serum. After centrifugation and careful removal of all supernatant fluid, 0.7 ml of the medium, which was 40% in polyethylene glycol (molecular weight range 950-1050) and 10% in dimethyl sulfoxide (Me2SO), was added over 10 sec with gentle stirring of the cells, following a modification of the procedure of Davidson et al. (15). After incubation for 30 sec at room temperature, 1 ml of medium was added over 1 min with gentle stirring. Additional (4 ml) medium was added over the succeeding 2 min. Then, 15 ml of medium was added at once and the suspension was centrifuged. The pellet was gently resuspended in medium supplemented with 10% fetal calf serum, and the suspension was incubated for 24 hr at 370C. After centrifugation and removal of the supernatant, the cells were then diluted into selective medium containing alanosine and adenine as the only purine source (4) and plated in 20 Falcon T 75 flasks. Hybrid populations that contained human chromosome 16 were then cloned in suspension.
Chromosomal Analysis. Three to four weeks following fusion, populations of cells were screened fot the presence of intact human chromosomes by Giemsa/trypsin banding-Hoechst 33258 sequential staining, as described (2, 3, 4, 16, 17), or by a second method in which simple Giemsa staining replaced the exposure to Giemsa/trypsin (4). CM-Cellulose Column Chromatography of Globin Chains. Hybrid populations (5 X 106 cells) incubated for 5 days in 1.25% Me2SO were exposed for 6 hr to 0.5 mCi (1 Ci = 3.7 X 1010 becquerels) of [3H]leucine (specific activity, 50 mCi/mmol) in 25 ml of medium. Fractionation of hemoglobin from other radioactively labeled cytoplasmic proteins and chromatography of the globin chains after heme extraction was carried out as described (4, 6). APRT Analysis. Cell pellets from stationary phase cultures were processed and analyzed for the presence of human and mouse ARPT as described by Tischfield and Ruddle (13). Analysis of Cytoplasmic RNA for Human Globin Coding Sequences. Cytoplasmic extracts were obtained from cultured cells as described (4). The RNA was purified by the method of Aviv and Leder (18). Human a globin mRNA, cytoplasmic poly(A)-containing RNA, and cDNA were prepared as described (4, 19). Human a globin cDNA was further purified by preparative hybridization and hydroxylapatite chromatography. First, an analytical scale Crot [initial concentration of RNA (moles/liter) X time (seconds)] analysis was performed by annealing a chain-enriched cDNA (80% pure) to a 4-fold excess of globin mRNA purified from reticulocytes of a donor with hemoglobin H disease (2). The resulting Crot curve showed transitions at CrOtj/2 (CrOt necessary for 50% hybridization) = 4.5 X 10-2 and 2.5 X 10-1 M X sec. The preparative reaction was performed with 3.6 ,ug of hemoglobin H mRNA (,B to a globin mRNA ratio = 4:1) and 20 ng of the a chain-enriched cDNA incubated in 750 ul of 50% formamide buffer (see below). This mixture was taken to a Crot value of 6 X 10-2 at 62°C, diluted with 7.5 ml of 50 mM sodium phosphate, pH 6.8/0.4% sodium dodecyl sulfate, and applied to a 0.7-ml bed volume hydroxylapatite column with 140 mM sodium phosphate, pH 6.8/0.4% sodium dodecyl sulfate. The hybrid was eluted with 480 mM sodium phosphate, pH 6.8/0.4% sodium dodecyl sulfate. cDNA was recovered as described (2). This method of purifying the a globin cDNA freed it of essentially
all cross-hybridizing human f3 globin coding sequences. The final product, [3H]cDNA, migrated as a single peak of radioactivity on 5% polyacrylamide gels in 99% formamide and had a molecular weight of 230,000 and a specific activity of 4.27 X 104 dpm/ng (1 dpm = 16.7 mBq). In the nucleic acid molecular hybridization assays, there were 2-jul aliquots of cDNA (about 0.1 ng) in 0.5 M NaCl/25 mM Hepes/0.5 mM EDTA/ 50% formamide (pH 6.8), which was used to dissolve appropriate amounts of salt-free, freeze-dried RNA. The mixtures were sealed in siliconized capillary tubes, denatured for 10 min at 950C, and then allowed to hybridize for 90 hr at 660C. Hybridization was terminated abruptly by plunging the capillaries into a dry ice/acetone bath. Hybrids were assayed as described (20) by their resistance to S1 nuclease. RESULTS
Chromosomal composition of hybrid cells derived by fusion of tetraploid mouse erythroleukemia cells and human peripheral blood leukocytes Previous work in our laboratory had shown that isolation of hybrid cells derived by fusion of mouse erythroleukemia cells deficient in APRT with human cells in selective medium supplemented with alanosine and adenine results in hybrid populations in which human chromosome 16 is present at a frequency of 80% (4). In the present study, mononuclear cells isolated from the peripheral blood of normal adult individuals by Ficoll-Hypaque centrifugation (14) were fused in the presence of polyethylene glycol and Me2SO with APRT-deficient mouse erythroleukemia cells that contained a near tetraploid complement of mouse chromosomes, as shown in Table 1. Analysis of metaphase spreads of the two hybrid populations resulting from this fusion by the species-specific stains showed that the hybrids had retained a tetraploid complement of mouse chromosomes and less than 15% of the chromosomes present in the original human parent cells. An example of a single metaphase spread of hybrid population 1, which was stained by two different species-specific stains, is shown in Fig. 1. As
..,
16
*
.'
A~6
ve
eA
'
FIG. 1. Single metaphase spread of hybrid population 1. (Left) Exposed to Hoechst 33258, which stains the human chromosomes with a uniform intensity but stains mouse chromosomes more intensely near the centromeres and less intensely along the arms. (Right) The same metaphase spread was stained with Giemsa after exposure to trypsin, which generates banding specific for each human and mouse chromosome. Human chromosome 16 is indicated. A few human chromosomes in addition to human chromosome 16 that were found in this metaphase spread are identified by additional small black arrows.
Biochemistry:
Deisseroth and Hendrick
Proc. Natl. Acad. Sci. USA 76 (1979)
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Table 1. Properties of hybrid cells derived by fusion of near tetraploid mouse erythroleukemia cells with human peripheral leukocytes
Total mouse chromosomes per cell*
% cells containing human chromosome 2 Cell line 3 4 6 9 11 8 12 21 10 13 16 20 Unfused parent mouse erythro72 leukemia cell 0 0 0 0 0 0 0 0 0 0 0 0 0 Hybrid cell 1 81 10 45 5 (uncloned) 60 25 15 65 15 45 5 85 35 0 Hybrid cell 2 71 4 4 (uncloned) 33 0 0 0 0 0 0 4 90 0 19 * Thirty metaphase spreads of each uncloned hybrid population were analyzed for the composition of human chromosomes. Human chromosomes that are not listed were not observed in any of the metaphase spreads examined.
shown by the data in Table 1 and Fig. 1, nearly every metaphase spread analyzed contained human chromosome 16 (which bears the human a globin structural gene) in spite of the extensive loss of human chromosomes. The frequency of the other human chromosomes retained by these hybrid cells is also presented in Table 1. Independent confirmation of the presence of human chromosome 16 in these hybrid populations was established by electrophoresis of protein extracts of the hybrid cells and autoradiographic analysis of the electrophoretograms for APRT. This enzyme is normally constitutively synthesized and is known to be coded for by human chromosome 16 (13). As shown by the data presented in Table 1 and Fig. 2, hybrid cell populations 1 and 2, which contain human chromosome 16, also contained the human form of APRT. Analysis of cytoplasmic RNA of hybrid cells for the
presence of human a globin mRNA
Hybrid populations 1 and 2, parent mouse erythroleukemia cells, and a continuously proliferating human lymphocytic cell line, Molt-4 (21), were incubated in 1.25% Me2SO for 5 days. This treatment is known to result in induction of expression of the mouse globin genes in mouse erythroleukemia cells (22, 23). Cytoplasmic poly(A)-containing RNA was purified from the Me2SO-induced hybrid cells and incubated with highly purified human a globin cDNA. Hybridization between mouse a globin mRNA and human a globin cDNA does not occur under the conditions used in our experiments, as reported by us (4) and shown in Fig. 3. The data presented in Fig. 3 also show that the cytoplasmic poly(A)-containing RNA of hybrid population 1 contained human a globin coding sequences after induction Origins
1
2
3
4
5
80 70 °- 60 *550
FIG. 2. Autoradiograph of a polyacrylamide slab gel for detection of human and mouse APRT in hybrid cell extracts. Extracts of mouse fibroblasts (lane 1), APRT-deficient mouse erythroleukemia cells (lane 3), hybrid cell 1 (lane 4), and human lymphocytes (lane 6) were subjected to electrophoresis and then exposed to the APRT reaction mixture. Lane 2 contained bromophenol blue and all other lanes (except for lane 5) contained approximately 10 ,g of protein.
0 40
+
28
+
0
1
C
C
0
40t
.C30 R 20 10 1 2 3 4 5 6*
300 0
Human APRT
in Me2SO. One can conclude from the ratio of the two abscissae (a to 0) in Fig. 3 that human a globin mRNA represented about 1 in 300 cytoplasmic poly(A)-containing RNA sequences in the hybrid cells. Also presented in Fig. 3 are control hybridization assays which included the incubation of human a globin cDNA under the same conditions with cytoplasmic poly(A)-containing RNA from the Me2SO-induced parent tetraploid mouse erythroleukemia cells and from Molt-4 after incubation with Me2SO. There was no cross hybridization of mouse erythroleukemia poly(A)-containing RNA with human (v globin cDNA under the conditions employed for the experiments. From our control assays, we know that the poly(A)containing RNA from the Me2SO-induced mouse parent cells contains about 1% mouse globin mRNA sequences. As shown in Fig. 3, there were no detectable human a globin coding sequences in the poly(A)-containing RNA from the human lymphocytic cell line Molt-4 at RNA to cDNA ratios up to 5000. When poly(A)-containing RNA from Molt-4 cells was used to extend the RNA to cDNA ratios up to 1.5 X 105, we still were unable to detect any human (v globin coding sequences in the RNA from these Me2SO-induced human lymphocytic cells
6
Q Co
22
900 1500
0
2500 5000 AD RNA/cDNA FIG. 3. Hybridization of human a globin cDNA to cytoplasmic RNA extracted from Me2SO-induced hybrid cell 1, which contained a near tetraploid complement of mouse chromosomes and human chromosome 16. Each 2-Ml incubation mixture contained 0.1 ng of human a globin cDNA and was incubated for 90 hr at 660C. The ratio of RNA to cDNA in each incubation is plotted versus the percentage of cDNA that formed a hybrid with RNA. Hybrid formation was assayed by resistance to S1 nuclease. In addition to human a globin cDNA, the incubation mixture contained: 0, human a globin mRNA; 0, cytoplasmic poly(A)-containing RNA from hybrid cells; 03, cytoplasmic poly(A)-containing RNA from parent mouse erythroleukemia cells; A, cytoplasmic poly(A)-containing RNA from Molt-4, a human
lymphocyte cell line.
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Proc. Natl. Acad. Sci. USA 76 (1979)
(unpublished results). Thus, transfer of the human a globin from nonerythroid donor cells, which were devoid of human globin coding sequences in cytoplasmic RNA, to tetraploid mouse erythroleukemia cells resulted in the activation of human globin gene expression. Study of human a globin chain synthesis in hybrid
genes
a
a
cells
The two hybrid tetraploid mouse erythroleukemia cell populations that contained human chromosome 16 were analyzed for the presence of human a globin chains by exposure of the Me2SO-induced hybrid cells to [3H]leucine for 6 hr, as described in Methods and by us previously (4-6). After purification of labeled hemoglobin from the hybrid cells and extraction of heme, the resulting globin chains were fractionated on columns of CM-cellulose. As shown by the data presented in Fig. 4 and Table 2, both of these hybrid populations exhibited expression of human a globin chains as well as mouse a and # globin chains.
We then analyzed four subclones isolated from hybrid populations 1 and 2, which had been derived from four independent fusion events and which contained human chromosome 16 in 100% of the cells analyzed (see Table 2). In all four subclones, induction by Me2SO resulted in the appearence of human a globin chains in addition to mouse a and globin chains. Thus, the hybrid cells and their subclones, which retained a tetraploid complement of mouse erythroleukemia chromosomes as well as human chromosome 16, reproducibly exhibit phenotypic expression, through the level of globin chain synthesis, of the human a globin genes derived from nonerythroid cells.
DISCUSSION The experiments summarized in this report have documented that the mechanisms responsible for the restriction of expression of human globin genes in nonerythroid cells are reversible by transfer of these genes into a mouse erythroleukemia cell in which a near tetraploid complement of mouse chromosomes has been retained. In addition, the correlation in the cell hybrids reported in this study between the production of human globin chains and the presence of human chromosome 16 confirms recent publications from our laboratory (2, 4) in which a
0
50
100 Fraction
150
FIG. 4. CM-cellulose chromatography of [3H]leucine-labeled globin chains in cell hybrid 1. This hybrid cell was derived by fusion of APRT-deficient mouse erythroleukemia cells with human peripheral blood leukocytes in which retention of a near tetraploid complement of mouse chromosomes as well as human chromosome 16 had occurred. A 6-hr labeling period was conducted after 5 days of incubation in Me2SO.
Table 2. Content of human a globin genes and human chromosome 16 in subclones of hybrid mouse erythroleukemia cells Cells positive Human a globin for human Hybrid chromosome 16* chains
Hybrid 1: Uncloned Clone 9 Clone 10
85 100 100
+ + +
Hybrid 2: Uncloned Clone 1 Clone 3
90 100 100
+ + +
* Twenty metaphase spreads of each hybrid clone and 30 metaphase spreads of each uncloned hybrid population were analyzed for human chromosome content.
the localization of the human a structural genes to this chromosome was established by independent means. The population used as the donor for the human globin structural genes was obtained from human peripheral blood by Ficoll-Hypaque centrifugation. None of the 50,000 cells studied was positive when stained for hemoglobin with benzidine, and the frequency of committed erythroid precursor cells cannot be placed above 1:5000 (24). Thus, the probability that the donor cells for the four independent fusion events derived from hybrid populations 1 and 2 were all of erythroid origin is less than 1 X 10-14. Moreover, measurements by other workers have shown that populations of peripheral blood leukocytes drawn from normal individuals in a manner similar to that used in our studies do not contain detectable levels of human globin mRNA (25). Studies by Miller et al. (26) and studies in our own laboratory (see Fig. 3) have shown that established cell lines exhibiting markers of lymphoid or myeloid (unpublished results) origin do not contain globin coding sequences in cytoplasmic RNA even after incubation in Me2SO. All of these studies taken together have established that the human globin genes transferred to mouse erythroleukemia cells in this report were derived from donor cells in which there were no detectable levels of human globin mRNA or globin chains. Miller et al. (26) have shown that lymphoblast cell lines in which there are no detectable levels of human globin mRNA do share a property in common with erythroid cells-that of complete sensitivity of the globin genes to digestion with DNase 1. Thus, the genetic mechanisms governing restriction of phenotypic expression of globin genes in nonerythroid hematopoietic cells must not rest on the configuration of the globin genes in so far as it is measurable by DNase 1 sensitivity. As discussed above, previous work with cell hybrids suggested that fusion of a diploid differentiated cell with a diploid undifferentiated cell results in hybrid cells in which loss of phenotypic expression is observed (7-9). Experiments by Axelrod et al. (27) showed that maintenance of the phenotype of hemoglobin inducibility by Me2SO is observed in hybrid cells derived by fusion of near tetraploid mouse erythroleukemia cells with human fibroblasts. Hybrid cells isolated by the same group of workers (derived by fusion of human fibroblasts with mouse erythroleukemia cells), which retained a near tetraploid complement of mouse chromosomes as well as human chromosome 11, contained human ( globin coding sequences in the cytoplasmic RNA after induction by Me2SO (28). Even though the 3 and Ty globin structural genes have been shown to be closely linked on chromosome 11 (3, 29), these hybrid cells contained no detectable levels of human y globin coding sequences in their cytoplasmic RNA (28). No data was presented
Biochemistry:
Deisseroth and Hendrick
by these authors however to establish the presence or absence of human d globin chains (28). Thus, one is unablf to conclude from the work of these authors whether they had isolated hybrid cells in which activation of full phenotypic expression of human /3 globin genes from nonerythroid cells had occurred. It is not known at this time whether the different patterns of differentiated gene expression seen in the fusion experiments summarized in this discussion arise primarily from the ploidy of the recipient mouse erythroleukemia cell or depend upon the retention or loss of chromosomes of human origin, which may exert a negative regulatory effect on the expression of globin genes in nonerythroid cells. Our experiments as well as those cited above (28) do suggest however that the mechanisms that mediate the differential expression of the two non-a (/3 and y) globin structural genes in adult and fetal life depend on a genetic process that is different from that responsible for the restriction of expression of globin genes in nonerythroid cells. This appears to be the case, because transfer of human chromosomes 11 and 16 from nonerythroid human donor cells to tetraploid mouse erythroleukemia cells resulted in activation of expression of a and / but not Sy globin structural genes. The experiments of Young et al. (30), which showed that both the y and : globin structural genes are equally sensitive to the digestive action of DNase 1 in adult erythroid cells, were interpreted by the authors as consistent with regulation of expression of y and /3 globin genes at the transcriptional or posttranscriptional level. Wood et al. (31) have also presented evidence that posttranscriptional events are responsible for the differential expression at the phenotypic level of the human /3 and 6 globin genes in adult erythroid cells. The presence of a globin chains in the hybrid tetraploid mouse erythroleukemia cells we have isolated, which also contain human a globin genes derived from nonerythroid cells, decisively demonstrates that the process that mediates restriction of expression of human globin genes in nonerythroid cells is not irreversible. These data also establish that transfer of the chromosomes bearing the human globin genes from nonerythroid cells to the near tetraploid mouse erythroleukemia cells results in activation of phenotypic expression of these genes, as shown by the synthesis of human globin chains. Application of the technology of restriction endonuclease mapping and plasmid cloning (32-34) to the study of these populations of hybrid cells may contribute structural information that would help clarify the precise mechanisms responsible for these findings. Further study of the experimental activation of phenotypic expression of human globin genes derived from nonerythroid cells may also result in information that will be of use in developing treatments for disease states in man in which activation of nonexpressed globin genes in erythroid cells would be of potential benefit. The authors recognize the expert technical assistance of Mary Blenkush and Sylvon Von Der Pool. Avian myeloblastosis virus RNA-directed DNA polymerase was obtained through Program Resources and Logistics, Viral Oncology Program, National Cancer Institute. 1.
Deisseroth, A., Velez, R. & Nienhuis, A. (1976) Science 191,
1262-1263. 2. Deisseroth, A., Nienhuis, A., Turner, P., Velez, R., Anderson, W. F., Ruddle, F., Lawrence, J., Creagen, R. & Kucherlapati, R. (1977) Cell 12,205-218.
Proc. Natl. Acad. Sci. USA 76 (1979)
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3. Deisseroth, A., Nienhuis, A., Lawrence, J., Giles, R., Turner, P. & Ruddle, F. (1978) Proc. Natl. Acad. Sci. USA 75, 14561460. 4. Deisseroth, A. & Hendrick, D. (1978) Cell 15,55-63. 5. Deisseroth, A. & Hendrick, D. (1978) in Cellular and Molecular Regulation of Hemoglobin Switching, eds. Stamatoyannopoulos, G. & Nienhuis, A. (Grune & Stratton, New York), pp. 761777. 6. Deisseroth, A., Barker, J., Anderson, W. F. & Nienhuis, A. (1975) Proc. Natl. Acad. Sci. USA 72,2682-2686. 7. Davidson, R. L. (1974) in Somatic Cell Hybridization, eds. Davidson, R. L. & De La Cruz, F. (Raven, New York), pp. 137-150. 8. Deisseroth, A., Burk, R., Picciano, D., Minna, J., Anderson, W. F. & Nienhuis, A. (1975) Proc. Natl. Acad. Sci. USA 72, 11021106. 9. Deisseroth, A., Velez, R., Burk, R., Minna, J., Anderson, W. F. & Nienhuis, A. (1976) Somatic Cell Genet. 2,373-384. 10. Orkin, S., Harosi, R. & Leder, P. (1975) Proc. Natl. Acad. Sci. USA 72, 98-102. 11. Conscience, J. F., Ruddle, F., Skoultchi, A. & Darlington, G. (1977) Somatic Cell Genet. 3, 157-172. 12. Darlington, G., Bernhard, H. & Ruddle, F. (1974) Science 185, 859-862. 13. Tischfield, J. & Ruddle, F. (1974) Proc. Natl. Acad. Sci. USA 71, 45-49. 14. Boyum, A. (1968) Scand. J. Clin. Lab. Invest. 21, Suppl. 97, 77-89. 15. Davidson, R. L., O'Malley, K. & Wheeler, T. B. (1976) Somatic Cell Genet. 2, 271-280. 16. Kozak, C., Lawrence, J. & Ruddle, F. (1977) Exp. Cell Res. 105, 109-117.
17. Yoshida, M. C., Ikeuchi, R. & Sasake, M. (1975) Proc. Jpn. Acad. 51, 184-187. 18. Aviv, J. & Leder, P. (1972) Proc. Natl. Acad. Sci. USA 69, 1408-1412. 19. Forget, B. G., Housman, D., Benz, E. J. & MaCaffrey, R. P. (1975) Proc. Natl. Acad. Sci. USA 72,984-988. 20. Jackson, J., Tolstoshev, P., Williamson, R. & Hendrick, D. (1976) Nucleic Acids Res. 3, 2019-2026. 21. Minowada, J., Ohnuma, T. & Moore, G. (1972) J. Natl. Cancer Inst. 49, 891-894. 22. Friend, C., Scher, W., Holland, J. C. & Sato, T. (1971) Proc. Natl. Acad. Sci. USA 68,378-382. 23. Cooper, M. C., Levy, J., CantorJ. N., Marks, P. A. & Rifkind, R. A. (1974) Proc. Natl. Acad. Sci. USA 71,1677-1680. 24. Clarke, B. J. & Housman, D. (1977) Proc. Natl. Acad. Sci. USA 74, 1105-1109. 25. Gianni, A. M., Dalla, F. R., Polli, E., Merisio, I., Giglioni, B., Comi, P. & Ottolenghi, S. (1978) Nature (London) 274, 610-611. 26. Miller, D. N., Turner, P., Nienhuis, A., Axelrod, D. C. & Gopalakrishnan, T. V. (1978) Cell 15,511-521. 27. Axelrod, D. C., Gopalakrishnan, T. V., Willing, M. & Anderson, W. F. (1978) Somatic Cell Genet. 4, 157-168. 28. Anderson, W. F., Willing, M. D. & Nienhuis, A. (1978) Blood 52, Suppl. 1, p. 108. 29. Smith, D. H., Clegg, J. B., Weatherall, D. J. & Gilles, H. M. (1973) Nature (London) New Biol. 246, 184-188. 30. Young, N., Benz, E. J., Jr., Kantor, J. A., Kretschmer, P. & Nienhuis, A. (1978) Proc. Natl. Acad. Sci. USA 75, 58845888. 31. Wood, W. G., Old, J. M., Roberts, A. V. S., Clegg, J. B. & Weatherall, D. J. (1978) Cell 15, 437-446. 32. Mears, J. G., Ramirez, F., Leibowitz, D. & Bank, A. (1978) Cell 15, 15-23. 33. Embury, S. H., Dozy, A. M. M. & Kan, Y. W. (1978) Blood 52, Suppl. 1, p. 111. 34. Mulligan, R. C., Howard, B. H. & Berg, P. (1979) Nature (London) 277, 108-114.
