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Induction of erythroid differentiation by cytoplast fusion in mouse erythroleukemia (friend) cells
Experimental Cell Research 159 (1985) 224-234
Induction of Erythroid Differentiation by Cytoplast Fusion in Mouse Erythroleukemia (Friend) Cells TOSHIO WATANABE, SHINTARO N O M U R A and MICHIO OISHI Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo, 113 Japan
An intracellular activity, which is induced by dimethyl sulfoxide (DMSO) or hexamethylenebisacetamide (HMBA) and leads to erythroid differentiation in mouse Friend cells, was characterized by cell fusion between genetically marked intact cells and cytoplasts. For this, a procedure for rapid selection of cybrids was devised by sensitizing non-fused cells with oligomycin. We were able to demonstrate that cytoplasts derived from DMSO- (or HMBA)-treated cells trigger erythroid differentiation upon fusion with UV-irradiated cells. The activity in the cytoplasts remained only transiently and its induction was inhibited by biologically active phorbol esters or cycloheximide. The activity, however, was not induced in cytoplasts by directly treating them with DMSO (or HMBA). These results indicate that (1) the intracellular erythroid-inducing activity is located in cytoplasts, (2) it acts in trans and induces erythroid differentiation as a dominant factor and (3) its production requires de novo nuclear protein synthesis. The mechanisms of the induction of the intracellular activity and of how it triggers erythroid differentiation are discussed. © 1985 Academic Press, Inc.
Many cell lines are now available which undergo in vitro differentiation in responding to a variety of inducing agents. Among them, in vitro differentiation of mouse erythroleukemia (Friend) cells [1] has been studied extensively as a model not only for differentiation of hematopoietic cells but also for cellular differentiation in general. After exposure to the inducing agents including dimethyl sulfoxide (DMSO) [2], hexamethylenebisacetamide (HMBA) [3] or butyric acid [4, 5], Friend cells convert to the cells with characteristics of erythroid cells. Despite numerous experiments, however, the molecular mechanism of how these agents act on the cells and eventually the cells are committed to the differentiation is still unknown. In recent cell fusion experiments, we have demonstrated that the in vitro erythroid differentiation of Friend cells is a result of a synergistic action of two independent intracellular reactions [6]. One is derived from cessation or disturbance of DNA replication and the other is mediated by a trans-membrane reaction which is triggered by most of the inducing agents. We have further shown that, as a result of the latter membrane-mediated reaction triggered by DMSO (or HMBA), an intracellular erythroid inducing activity is induced at the very early stage after the treatment [7]. The activity is detected only by cell fusion with the cells which had been treated with DNA damaging agents, such as ultraviolet (UV) light. The induction process of the activity consists of at least two stages, the initial stage which is independent of metabolites in the medium but sensitive to Copyright © 1985 by Academic Press, Inc. All fights of reproduction in any form reserved 0014-4827/85 $03.00
Erythroid differentiation in Friend cells
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biologically active phorbol esters and the second stage which is coupled to cellular metabolic reactions, especially to protein synthesis. The induced activity remains transiently in the cells lasting for only a few hours. Although these experiments have provided some information concerning the mechanism of erythroid differentiation in Friend cells, they have raised several new questions about the nature of the intracellular activity which is induced after DMSO or HMBA treatment. Does the activity correspond to a defined molecule? Does it act in trans or in cis? Are nuclei of the treated cells involved in producing the activity? Some of these questions might be answered by experiments employing cell (cybrid) fusion between cells and cytoplasts prepared from the DMSO- or HMBA-treated cells. Use of cytoplasts would eliminate complications derived from the presence of nuclei in the treated cells. However, one of the difficulties in such experiments was to select cybrids in a short period of time (several days) in which erythroid differentiation is completed. Current procedures for cybrid selection are all designed for a long-term selection (over several weeks) such as for establishing a cell line with a newly introduced cytoplasmic element. In attempts to find a condition for rapid selection of cybrids, we have found that presence of oligomycin in the selection medium considerably accelerates the cybrid selection process, thus enabling us to select the cybrids before erythroid differentiation is completed. In this paper, we report that cytoplasts obtained fi'om DMSO- (or HMBA)-treated cells are able to trigger erythroid differentiation upon fusion with UV-irradiated cells. The activity exhibited a transient nature and its induction was blocked by biologically active phorbol esters or cycloheximide. On the other hand, the activity was not induced in cytoplasts by directly treating them with DMSO (or HMBA). Based upon these observations, the following points have become clear in respect to the mechanism of in vitro erythroid differentiation in mouse Friend cells. (1) The intracellular activity produced by DMSO (or HMBA) for erythroid differentiation exists in cytoplasts. (2) The activity acts in trans and induces erythroid differentiation as a dominant factor. (3) Although the activity exists in cytoplasts, a protein(s) of nuclear origin, plays a role in producing the activity.
MATERIALS AND METHODS Materials Thymidine, aminopterin, hypoxanthine were obtained from Sigma. Ouabain (g-strophanthin) and oligomycin were purchased from Boehringer Mannheim. Chloramphenicot was obtained from Sankyo. 12-O-Tetradecanoylphorbol 13-acetate (TPA) was obtained from Dr P. Borchert (Eden Prairie, Minn.). Phorbol was a gift from Drs M. Terada and T. Sugimura. Polyethylene glycol 6000 was purchased from J. T. Baker Chemical (Phillipsburg, N.J.). Hexamethylenebisacetamide (HMBA) was a gift from Dr T. Yamane. Rhodamine 123 was obtained from Eastman Kodak. Eagle's minimum essential medium (MEM, in powder) was purchased from Nissui Seiyaku (Tokyo). Fetal calf serum (FCS) and calf serum were obtained from Flow Lab and Nippon Biotest, respectively. All the reagents used were reagent grade. Exp Cell Res 159 (1985)
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Cells A Friend (murine erythroleukemia) cell line (DS 19) was obtained from Dr M. Terada. This cell line was derived from cell line 745A. A mutant of DS 19, designated as DS 19 (Oua~Cm s) in this paper, lacks functional thymidine kinase (Tk-) and was a gift from Dr R. A. Rifkind. We will not refer to the Tk- mutation in this paper, A double mutant of DS 19 that lacks thymidine kinase and is resistant to ouabain (3 raM), designated as IAM I01 (Oua"CmS), was isolated in this laboratory from N-methylN'-nitro-N-nitrosoguanidine-treated DS 19 (OuaSCm s) cells. These mutant cells are all chloramphenicol-sensitive (Cm~). A chloramphenicol-resistant Friend cells (Cm r, IAM 111) was constructed by introducing Cm r marker into Friend cells (DS 19) from a chloramphenicol-resistant mouse L cell line (B 82 Cm ~) by cytoplast fusion, followed by successive selections starting from low level to relatively high level chloramphenicol resistance (100 ~g/ml). The chloramphenicol-resistant mouse L cell line (13 82 Cm r) [8] was supplied by Dr T. Sekiguchi. The chloramphenicol-resistant Friend cells (IAM 111) exhibited a normal pattern of erythroid differentiation after exposure to DMSO, HMBA and other erythroidinducing agents in the presence or absence of chloramphenicol (80 ~tg/ml) (data not shown).
UV Irradiation Confluently grown cells (2x 106 cells/ml) were collected by centrifugation (500 g, 5 rain) at room temperature and resuspended in phosphate-buffered saline (PBS) at a final cell density of 5 X 1 0 6 cells/ml. The celt suspension (2 ml) was transferred to a plastic Petri dish (60x 12 mm) and irradiated under a Toshiba GL 15 (15 W) germicidal UV lamps at a distance of 52 cm, which gave UV intensity 1.52 J/mZ/sec. Under this condition, approx. 50 % (between 40-60 %) of the ceils survived when we examined their plating efficiency on methylcellulose-containing medium. After irradiation, the cells were collected by centrifugation and resuspended (8x 105 cells/ml) in MEM supplemented with FCS (12 %) for further incubation (24 h) at 37°C.
Preparation of Cytoplasts L cells (B 82 Cm ~) were enucleated according to the method of Wigler et al. [9], in which the cells were centrifuged through Ficoll density gradient in the presence of cytochalasin B. Friend cells were enucleated by the method of Ohara et al. [10]. This method consistently yielded approx. 40% of enucleation and gave a purity of the cytoplast over 99.9 %.
Cytoplast Fusion and Cybrid Selection Cytoplast fusion was performed according to a modification of Pontecorvo's procedure for cell fusion [1 l]. In essence, intact Friend cells (1 x 106 cells) and cytoplasts (3 x 10 6 cells) were first mixed and centrifuged (1 200 g, 5 min) at room temperature. The pellet was mixed with 0.2 ml of polyethylene glycol 6000 (50%, w/w, in water) and kept at room temperature for 2 rain. MEM (1 ml) was then added and, after gentle mixing, the sample was left at room temperature for 3 rain. For cybrid selection, the cells were then diluted with 4 ml of MEM supplemented with calf serum (10%), centrifuged (500 g, 5 rain), and resuspended in 1 ml of MEM containing FCS (12 %), chloramphenicol (80 Ixg/ml) and ouabain (3 raM). After 4 days incubation at 37°C in an incubator with 5 % CO2, oligomycin was added to the selection medium at a final concentration of 10 ng/ml and the cells were incubated for one more day.
Fluorescent Staining Rhodamine 123 staining of the cells was carried out as described by Johnson et al. [12]. Microscopy and photography were performed with a Olympus (BH2-RFK) fluorescence microscope. All photographs were taken using Fujicolor HR 400 film.
Cell Fixation on Polylysine-Coated Coverglass Two or three drops of polylysine solution (1 mg/ml) were placed on a coverglass. After 5 min, the covergtass was washed twice with double-distilled water and dried at room temperature. Cell
Exp CellRes i59 (1985)
Erythroid differentiation in Friend cells 227
Fig. 1. Selection of cybrids between intact cells and rhodamine .t23-1abelled cytoplasts. ][AM 111
(OuaSCm~) cells were labelled with rhodamine 123. Cytoplasts prepared from these cells were fused with IAM 101 (OuarCmS). After 4 days' incubation with chloramphenicol (80 Ixg/ml) and ouabain (3 Exp Cell Res 159 (1985)
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suspension (one or two drops) was placed on the polylysine-coated coverglass and incubated for 2 h at 37°C in an incubator with 5 % COz. After removing the medium and dead cells by aspiration, the coverglasses were rinsed with fresh medium.
RESULTS
Rapid Selection of Cybrids by Sensitization of Non-Fused Cells with Oligomycin In order to characterize the intracellular erythroid inducing activity by cytoplast fusion, we first explored conditions for cybrid selection. In early experiments, cytoplasts (enucleated cells), which were prepared from chloramphenicolresistant (Cm ~) Friend cells (IAM 111), were fused with intact cells (IAM 101) with a nuclear genetic marker (ouabain resistance, Oua r) and the samples were incubated in a selection medium containing chloramphenicol (80 ~tg/ml) and ouabain (3 mM/ml). While a small portion (1%) of the cells which were presumably originated from fused cells (cybrids) between the intact cells (OuarCm s) and the cytoplasts (OuaSCmr) continued to grow in the selection medium, the majority of the cells which consisted of non-fused cells did not lose their viability after 5 days' incubation in the selection medium (data not shown). Since in vitro erythroid differentiation in Friend cells is generally completed within 5 days, it was obvious that selection of cybrids by chloramphenicol and ouabain alone was inadequate for analysing the role of cytoplasmic elements in erythroid differentiation. We suspected that the sustained resistance to chloramphenicol of the nonfused cells was due to residual mitochondrial proteins which are still able to support minimum mitochondrial functions. Accordingly, we employed oligomycin, an inhibitor for oxidative phosphorylation, and found that it greatly accelerated killing of the non-fused cells. For example, when oligomycin (10 ng/ml) was added to the selection medium after 4 days incubation with chloramphenicol (80 ~tg/ml) and ouabain (3 mM), the majority of the cells were killed within 24 h. As seen in table 1, while all the cells remained trypan blue-negative when the cells were incubated with chloramphenicol (and ouabain) alone, addition of oligomycin after 4 days' incubation with chloramphenicol and ouabain converted essentially all the cells to trypan blue-positive cells which consisted of more than 99.9 % of non-fused cells. On the other hand, there was a small but significant difference in the number of trypan blue-negative cells between the fused cells with Cm s cytoplasts and Cm r cytoplasts, indicating selection of cybrids with Cm r cytoplasraM), oligomycin (10 ng/ml) was added to the medium and the cells were incubated for one more day. The cells were then fixed on coverslips coated with polylysine and photographs were taken through a fluorescent microscope (Olympus BH2-RFK). (A) Without; (B) with oligomycin. In order to show a large number of rhodamine-positive cells, the oligomycin-treated samples (B) were concentrated (approx, 100-fold by centrifugation) before applying them to polylysine-coated coverglass slips. The dead cells are not fixed on the slips under this condition. For details, see Materials and Methods. Exp Cell Res 159 (1985)
..... ~- grythroid differentiation in Friend cells 229 Table 1. Cybrid selection by oligomycin Treatment Cytoplast fusion
Cells
Cytoplasts prepared from
IAM 101 (OuarCm s) x IAM 101 (OuarCmS)× DS 19 (Oua~Cm ~) IAM101 (OuarCm~)× I A M I l l (OuaSCm ~)
- Oligomycin
+ Oligomycin
TB ÷
TB-
TB +
TB-
0 0 0
- 1 x 104 - 1 x 104 ~1×104
- 1 x 104 - 1 × 104 ~1×104
3 3 110
Friend cells (IAM 101 OuarCm s) were fused with cytoplasts prepared from either IAM 101 (Oua~Cm s) or IAM 111 (OuaSCm r) as described in Materials and Methods. After 4 days' incubation with chloramphenicol (80 ~tg/ml) and ouabain (3 mM), the samples were further incubated for 1 day with or without oligomycin (10 ng/ml) in addition to chloramphenicol and ouabain. The cells were then stained with trypan blue [0.01% (w/w)]. Trypan blue positive (TB ÷) and negative (TB-) cells (total approx. 104 cells) were counted under a microscope (Olympus BH2-RFK). For details, see Materials and Methods.
mic marker (but not with Cm s marker) under this condition (table 1). From the number of the trypan blue-negative cells appeared from the cell fusion with Cm r cytoplasts, we estimated that approx. 1% of the original cells formed cybrids as a result of fusion with the cytoplasts. The effect of oligomycin on cybrid selection was further confirmed by cell fusion experiments with cytoplasts marked by rhodamine 123 which specifically stains mitochondria. Cytoplasts were first stained with rhodamine 123 and, after their fusion and selection in both the absence and presence of oligomycin in addition to chloramphenicol and ouabain, the samples were examined under a
Table 2. Selection o f cybrids between intact cells and rhodamine 123-labelled cytoplasts--a quantitative analysis
Source of cytoplasts -
DS 19 (Oua~Cm s) IAM 111 (Oua~Cm r)
- Oligomycin
+ Oligomycin
Rho ÷
Rho +
Rho-
Rho-
0
~ 2 × 104
0
0
140 146
~ 2 × 104 ~ 2 x 104
0 101
0 9
DS 19 (Oua~Cm s) and IAM 111 (OuaSCm ~) cells were labelled with rhodamine 123 by the method of Johnson et al. [12]. Cytoplasts prepared from these cells were fused with IAM 101 (OuarCmS). After cybrid selection in the presence or absence of oligomycin (10 ng/ml) as described in Materials and Methods, the cells were fixed on coverglass slips coated with polylysine. Numbers of rhodaminepositive (Rho ÷) and -negative (Rho-) cells (the initial number of the cells; approx. 2x 104 cells) were counted under a fluorescence microscope (Olympus BH2-RFK). The drastic reduction in R h o - cells in oligomycin-treated sample was due to the loss of dead cells (non-fused cells) while washing the samples on polylysine-coated coverglass slips. For details, see Materials and Methods. Exp Cell Res 159 (1985)
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Table 3. Effect of various compounds on the induction of erythroid-inducing
activity in cytoplasts B ÷ cells (%) Treatment (donor)
Recipient cells - UV + UV
None
0.4
2.5
DMSO HMBA DMSO+TPA DMSO+Phorbol DMSO+Cycloheximide Control (without cell fusion)
1.2 0 0.8 1.0 0.4 0
8.5 6.9 1.4 6.4 0.9 0.4
Friend cells, IAM 111 (Oua~Cmr), were grown as described in Materials and Methods. The cells were then diluted (1 : 1) with a fresh medium and treated with various compounds. After 6 h, the cells were enucleated, fused with IAM 101 (OuarCm S) cells which had been irradiated with UV light (20 J/m 2) 24 h earlier. After cytoplast fusion, the cells were incubated in the presence of chloramphenicol (80 ~tg/ml) and ouabain (3 mM) for 4 days. Oligomycin (10 ng/ml) was then added to the medium and the cells were incubated for one more day. Benzidine-positive (B ÷) cells were then scored. Total of approx. 3x 103 cells were scored for each sample. Concentrations of the compounds used; DMSO 1.8% (v/v), HMBA 5 mM, TPA 100 ng/ml, phorbol 100 ng/ml and cycloheximide 10 ~tg/ml. For the control (without cell fusion) experiment, the cells (IAM I01), with or without UV irradiation, were incubated in the medium (without chloramphenicol and oligomycin) for 5 days and benzidine-positive (B ÷) cells were scored.
f l u o r e s c e n c e m i c r o s c o p e . A typical p i c t u r e o f the c y b r i d s o b t a i n e d with rhodamine 123-stained c y t o p l a s t s ( w i t h o u t o l i g o m y c i n ) is s h o w n in fig. 1 A. As seen in fig. 1 B, addition o f o l i g o m y c i n in the selection m e d i u m essentially eliminated all the n o n - s t a i n e d cells w h i c h c o n s i s t e d o f n o n - f u s e d F r i e n d cells. In table 2, we s h o w the results o f a quantitative analysis o f the e x p e r i m e n t s . It is quite clear that r h o d a m i n e 123-positive cells s u r v i v e d in the selection m e d i u m o n l y w h e n the cell fusion was p e r f o r m e d with C m r c y t o p l a s t s . F u r t h e r m o r e , the e x p e r i m e n t s indicate that the surviving cells in the p r e s e n c e o f c h l o r a m p h e n i c o l (and ouabain) and o l i g o m y c i n were, in fact, derived f r o m the c y b r i d s f o r m e d b e t w e e n intact cells and c y t o p l a s t s .
Characterization of lntracellular Erythroid-Inducing Activity Produced by Erythroid-Inducing Agents B y e m p l o y i n g this rapid c y b r i d selection, we c h a r a c t e r i z e d the intracellular activity i n d u c e d b y typical e r y t h r o i d - i n d u c i n g agents. T h e cells ( I A M 111 OuaSCm r) were i n c u b a t e d with D M S O ( 1 . 8 % , v/v) and, at different time intervals, c y t o p l a s t s were p r e p a r e d f r o m the cells and f u s e d with the cells ( I A M 101 O u a r C m s) w h i c h h a d b e e n irradiated with U V light 24 h before. T h e cells were then s u b j e c t e d to the c y b r i d selection, 4 d a y s ' i n c u b a t i o n with c h l o r a m p h e n i c o l Exp Cell Res 159 (1985)
Erythroid differentiation in Friend cells
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Fig. 2. Induction of intracellular erythroid-inducing activity in cytoplasts by DMSO. Friend cells, IAM 111 (OuaSCmr), were grown in MEM supplemented with FCS (12 %) at 37°C to a cell density of 2x106 cells/ml. The cells were then diluted (1 : 1) with the same medium and DMSO was added to a final concentration of 1.8 % (v/v). At different time intervals, the cells were enucleated and cytoplasts were fused with IAM 101 (OuarCm s) cells which had been irradiated by UV light (20 J/m 2) 24 h before. After 4 days incubation with chloramphenicol (80 ~tg/ml) and ouabain (3 raM), oligomycin (10 ng/ml) was added to the medium and the cells were incubated for one more day. Benzidine-positive (B ÷) cells were then scored. Cytoplasts from DMSO-treated cells; fused with O, UV irradiated cells; I , control (-UV) cells. Cytoplasts from control cells; fused with ©, UV-irradiated cells; [], control (-UV) cells. A total of approx. 3 x 103 cells were scored for each sample. For details, see Materials and Methods. Fig. 3. Absence of erythroid-inducing activity in cytoplasts directly treated with DMSO. Friend cells, IAM 111 (OuaSCm~), were grown in MEM supplemented with FCS (12 %) at 37°C to a cell density of 2x 106 cells/ml. The cells were then enucleated. The cytoplasts were suspended in the same medium (1 x l06 cytoplasts/ml) and exposed to 1.8 % (v/v) DMSO for various length of periods. At different intervals as shown in the figure, the cytoplasts were withdrawn, washed once with MEM and mixed with IAM 101 cells (OuarCm s, 1 × 106 cells) which had been irradiated by UV light (20 J/m 2) at 24 h before. After cytoplast fusion, the samples were incubated with chloramphenicol (80 ~tg/ml) and ouabain (3 mM) for 4 days. Oligomycin (10 ng/ml) was then added to the medium and the cells were incubated for one more day. Benzidine-positive (B ÷) cells were then scored. A total of approx. 3 x 103 cells were scored for each sample. For details, see Materials and Methods.
(80 ~tg/ml) and ouabain (3 mM), followed by 1 day incubation with oligomycin (10 ng/ml) in addition to chloramphenicol and ouabain. Cells with accumulated hemoglobin, a characteristic of erythroid cells, were then scored among surviving cells by benzidine staining [13]. As seen in fig. 2, the cytoplasts prepared from DMSO-treated cells were able to induce erythroid differentiation upon fusion with UV-irradiated cells. The extent of the induction (8.5-10% in seven independent experiments) was somewhat lower than that by cell fusion between intact cells, in which 15-20% of the cells became benzidine-positive. As shown in fig. 2, the induction of the activity was not seen upon fusion with control (non-UV-irradiated) cells. These results are consistent with the results of the previous cell fusion experiments that erythroid differentiation in Friend cells is a synergistic result of two distinctive intracellular reactions [6, 7]. Furthermore these results suggest that the intracellular activity Exp Cell Res 159 (1985)
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Watanabe, Nomura and Oishi
induced by inducing agents exists in cytoplasts, acts in trans and triggers erythroid differentiation as a dominant factor. As also shown in fig. 2, the induction of the activity exhibited a transient nature, although DMSO was continuously present in the medium until the time of preparation of the cytoplasts. The activity in the cytoplasts reached the maximum at 6 h after addition of the inducing agent (DMSO) and declined thereafter. A similar transient nature of the activity had been demonstrated in the cell fusion between two intact Friend cells [7]. The nature of the DMSO-induced activity in cytoplasts was further investigated by examining the effect of biologically active phorbol esters and cycloheximide. We had previously shown that the induction of the erythroid-inducing activity by DMSO (or HMBA) is inhibited by biologically active phorbol esters or cycloheximide. Friend cells (IAM 111 QuaSCmr) were incubated with DMSO together with a typical biologically active phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), or cycloheximide. Cytoplasts prepared from the treated cells were then fused with UV-irradiated or non-irradiated cells and benzidinepositive cells were scored after the cybrid selection. As seen in table 3, it is quite clear that the induction of the activity in the cytoplasts was inhibited by TPA and cycloheximide. Phorbol, a derivative of TPA without a tumor-promoting activity had little effect. We also show that HMBA, another potent erythroid inducer, induced the activity as DMSO did. These results are essentially the same as obtained with previous cell fusion experiments employing intact cells [6, 7], indicating that the erythroid-inducing activity induced in the cytoplasts is the same as that induced in intact cells. As described above, the induction of the erythroid-inducing activity in cytoplasts is inhibited by cycloheximide. This strongly suggests that a protein(s) of nuclear origin (vs mitochondrial origin) is involved in the induction of the activity in the cytoplasts, although the activity remains in the cytoplasts. Probably a signal produced by DMSO or HMBA through a TPA-sensitive trans-membrane reaction reaches to the nuclei at first place. A gene(s) is then activated in responding to the signal and its product(s) is transferred to cytoplasts to express its erythroid-inducing activity. Alternatively, induction of a protein of nuclear origin may be required for the activation of an erythroid-inducing factor which is constitutively present in cytoplasts. In order to further confirm the involvement of nuclei in inducing the activity, we conducted an experiment in which isolated cytoplasts were directly incubated with DMSO and then fused with UV-irradiated cells. As seen in fig. 3, the erythroid-inducing activity was not developed when cytoplasts were directly treated with DMSO, confirming that nuclei play a role in producing the erythroid-inducing activity in cytoplasts. The lack of induction of the activity in the isolated cytoplasts was not due to loss of fundamental physiological functions in the cytoplasts during incubation with DMSO, for the cytoplasts thus treated still maintained at least 80 % of capacity to rescue chloramphenicol sensitive cells after cytoplast fusion (data not shown). Exp Cell Res 159 (1985)
Erythroid differentiation in Friend cells
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DISCUSSION Use of a new rapid selection procedure for cybrids has enabled us to conduct a series of experiments in which erythroid differentiation can be induced by cell (cybrid) fusion with cytoplasts prepared from the cells treated with typical erythroid inducing agents, such as DMSO or HMBA. We showed that cytoplasts thus prepared still maintain an activity which is able to trigger erythroid differentiation upon fusion with UV-irradiated cells. These results indicate that the induced activity, whatever its molecular nature is, is located in cytoplasts and one can trap it in isolated cytoplasts. Furthermore, it has become clear that the activity, as a dominant factor, triggers erythroid induction by a trans-acting manner, provided that the recipient cells had been irradiated by UV light. We further showed that, although the erythroid-inducing activity is located in cytoplasts, the induction is inhibited by cycloheximide. Obviously, nuclei play a role in producing the activity probably through synthesis of a specific protein(s). What is the molecular basis for the activity in the cytoplasts? From the experiments described above alone, it is difficult to determine whether the induced activity resides in cytoplasmic membrane or cytoplasm. If the former is the case, Friend cells may be differentiated into erythroid cells by even partial alteration of their membrane structure caused by the integration of the modified membrane derived from the DMSO (or HMBA)-treated cells. On the other hand, if a cytoplasmic factor is responsible for the activity, the activity may correspond to a defined molecule(s), whose identification, of course, remains to be a subject of future studies. In any event, the activity induced by erythroid-inducing agents acts in trans and seems to be dominant over the pre-existing activity which keeps the cells from committing to erythroid differentiation. Consistent with the previous observations [7], we have shown that cycloheximide blocks the induction of the activity in the cytoplasts, indicating that a protein of nuclear origin is required in inducing the activity. Apparently, a signal produced by a trans-membrane reaction reaches nuclei at first place and then alters expression of a specific gene(s). Probably either the gene product itself corresponds to the activity or it indirectly affects the pre-existing activity in the cytoplasts. Recently, we have identified a novel nuclear protein (p54) in Friend cells whose synthesis is induced by inhibitors for erythroid differentiation, such as biologically active phorbol esters [14]. Constitutive as well as induced synthesis of p54 is inhibited by erythroid inducing agents including DMSO, HMBA and actinomycin D. These results suggest that p54 plays a role in erythroid differentiation in Friend cells as a negatively controlling element. A protein like p54 may be a repressor for the expression of this putative nuclear gene which is responsible for the induction of the erythroid-inducing activity in cytoplasts. There are several reports concerning the role of trans-acting cellular factors in erythroid differentiation [15-18]. Most of such works were carried out with hybrid cells produced by cell fusion between erythroid cells and non-erythroid Exp Cell Res 159 (1985)
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Watanabe, N o m u r a and Oishi
cells. T h e s e r e p o r t s i n d i c a t e that e r y t h r o i d - s p e c i f i c g e n e s are a c t i v a t e d in the h y b r i d cells s u g g e s t i n g the p r e s e n c e o f a trans-acting factor(s) in the d i f f e r e n t i a t e d ceils. A m o n g t h e m , R N A p o l y m e r a s e II was i m p l i c a t e d as o n e o f c r u c i a l c e l l u l a r factors for s u c h a n a c t i v a t i o n [17]o A t the p r e s e n t m o m e n t , the r e l a t i o n s h i p b e t w e e n t h e s e trans-acting c y t o p l a s m i c f a c t o r s a n d the p u t a t i v e f a c t o r d i s c u s s e d in the p a p e r is n o t c l e a r a n d m u s t wait for f u r t h e r i n v e s t i g a t i o n . We are grateful to Ms MJyuki Harada for her assistance in preparing the manuscript. We thank Dr Toyozo Sekiguchi for kindly providing us a chloramphenicol-resistant mouse L cell line used for constructing the Friend cell (IAM 111) and for his valuable suggestions during the course of the study. We also thank Dr Taihei Nozawa for his help in taking pictures of cybrids. This work was supported by Grant-in-Aid for special project research, Cancer-Bioscience, from the Ministry of Education, Science and Culture of Japan.
REFERENCES 1. Friend, C, Patuleia, M C & deHarven, E, Natl cancer inst monogr 228 (1966) 505. 2. Friend, C, Scher, W, Holland, J G & Sato, T, Proc natl acad sci US 68 (1971) 378. 3. Reuben, R C, Wife, R L, Breslow, R, Rifkind, R A & Marks, P A, Proc natl acad sci US 73 (1976) 862. 4. Leder, A & Leder, P, Cell 5 (1975) 319. 5. Takahashi, E, Yamada, M, Saito, M, Kuboyama, M & Ogasa, K, Gann 66 (1975) 577. 6. Nomura, S & Oishi, M, Proc natl acad sci US 80 (1983) 210. 7. Kaneko, T, Nomura, S & Oishi, M, Cancer res 44 (1984) 1756. 8. Sekiguchi, T, Tosu, M, Yoshida, M C, Oikawa, A, Ishihara, K, Fujiki, H, Tumuraya, M & Kameya, T, Somatic cell genetics 8 (1982) 605. 9. Wigler, M H & Weinstein, I B, Biochem biophys res commun 63 (1975) 669. 10. Ohara, J, Sekiguchi, T & Watanabe, T, J immunol methods 45 (1981) 239. I1. Pontecorvo, G, Somatic cell genet 1 (1975) 397. 12. Johnson, L V, Walsh, M L & Chen, L B, Proc natt acad sci US 77 (1980) 990~ 13. Orkin, S H, Harosi, F I & Leder, P, Proc natl acad sci US 72 (1975) 98. 14. Mitsuse, S & Oishi, M. Submitted for publication. 15. Deisseroth, A & Hendrick, D, Cell 15 (1978) 55. 16. Willing, M C, Nienhnis, A W & Anderson, W F, Nature 277 (1979) 534. 17. Zuckerman, S H, Linder, S & Ringertz, N R, J cell physiol 113 (1982) 99. 18. Chao, M V, Mellon, P, Charnay, P, Maniatis, T & Axel, R, Cell 32 (1983) 483. Received September 27, 1984 Revised version received February 8, 1985
Exp CellRes [59 (1985)
Printedin Sweden
Induction of Erythroid Differentiation by Cytoplast Fusion in Mouse Erythroleukemia (Friend) Cells TOSHIO WATANABE, SHINTARO N O M U R A and MICHIO OISHI Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo, 113 Japan
An intracellular activity, which is induced by dimethyl sulfoxide (DMSO) or hexamethylenebisacetamide (HMBA) and leads to erythroid differentiation in mouse Friend cells, was characterized by cell fusion between genetically marked intact cells and cytoplasts. For this, a procedure for rapid selection of cybrids was devised by sensitizing non-fused cells with oligomycin. We were able to demonstrate that cytoplasts derived from DMSO- (or HMBA)-treated cells trigger erythroid differentiation upon fusion with UV-irradiated cells. The activity in the cytoplasts remained only transiently and its induction was inhibited by biologically active phorbol esters or cycloheximide. The activity, however, was not induced in cytoplasts by directly treating them with DMSO (or HMBA). These results indicate that (1) the intracellular erythroid-inducing activity is located in cytoplasts, (2) it acts in trans and induces erythroid differentiation as a dominant factor and (3) its production requires de novo nuclear protein synthesis. The mechanisms of the induction of the intracellular activity and of how it triggers erythroid differentiation are discussed. © 1985 Academic Press, Inc.
Many cell lines are now available which undergo in vitro differentiation in responding to a variety of inducing agents. Among them, in vitro differentiation of mouse erythroleukemia (Friend) cells [1] has been studied extensively as a model not only for differentiation of hematopoietic cells but also for cellular differentiation in general. After exposure to the inducing agents including dimethyl sulfoxide (DMSO) [2], hexamethylenebisacetamide (HMBA) [3] or butyric acid [4, 5], Friend cells convert to the cells with characteristics of erythroid cells. Despite numerous experiments, however, the molecular mechanism of how these agents act on the cells and eventually the cells are committed to the differentiation is still unknown. In recent cell fusion experiments, we have demonstrated that the in vitro erythroid differentiation of Friend cells is a result of a synergistic action of two independent intracellular reactions [6]. One is derived from cessation or disturbance of DNA replication and the other is mediated by a trans-membrane reaction which is triggered by most of the inducing agents. We have further shown that, as a result of the latter membrane-mediated reaction triggered by DMSO (or HMBA), an intracellular erythroid inducing activity is induced at the very early stage after the treatment [7]. The activity is detected only by cell fusion with the cells which had been treated with DNA damaging agents, such as ultraviolet (UV) light. The induction process of the activity consists of at least two stages, the initial stage which is independent of metabolites in the medium but sensitive to Copyright © 1985 by Academic Press, Inc. All fights of reproduction in any form reserved 0014-4827/85 $03.00
Erythroid differentiation in Friend cells
225
biologically active phorbol esters and the second stage which is coupled to cellular metabolic reactions, especially to protein synthesis. The induced activity remains transiently in the cells lasting for only a few hours. Although these experiments have provided some information concerning the mechanism of erythroid differentiation in Friend cells, they have raised several new questions about the nature of the intracellular activity which is induced after DMSO or HMBA treatment. Does the activity correspond to a defined molecule? Does it act in trans or in cis? Are nuclei of the treated cells involved in producing the activity? Some of these questions might be answered by experiments employing cell (cybrid) fusion between cells and cytoplasts prepared from the DMSO- or HMBA-treated cells. Use of cytoplasts would eliminate complications derived from the presence of nuclei in the treated cells. However, one of the difficulties in such experiments was to select cybrids in a short period of time (several days) in which erythroid differentiation is completed. Current procedures for cybrid selection are all designed for a long-term selection (over several weeks) such as for establishing a cell line with a newly introduced cytoplasmic element. In attempts to find a condition for rapid selection of cybrids, we have found that presence of oligomycin in the selection medium considerably accelerates the cybrid selection process, thus enabling us to select the cybrids before erythroid differentiation is completed. In this paper, we report that cytoplasts obtained fi'om DMSO- (or HMBA)-treated cells are able to trigger erythroid differentiation upon fusion with UV-irradiated cells. The activity exhibited a transient nature and its induction was blocked by biologically active phorbol esters or cycloheximide. On the other hand, the activity was not induced in cytoplasts by directly treating them with DMSO (or HMBA). Based upon these observations, the following points have become clear in respect to the mechanism of in vitro erythroid differentiation in mouse Friend cells. (1) The intracellular activity produced by DMSO (or HMBA) for erythroid differentiation exists in cytoplasts. (2) The activity acts in trans and induces erythroid differentiation as a dominant factor. (3) Although the activity exists in cytoplasts, a protein(s) of nuclear origin, plays a role in producing the activity.
MATERIALS AND METHODS Materials Thymidine, aminopterin, hypoxanthine were obtained from Sigma. Ouabain (g-strophanthin) and oligomycin were purchased from Boehringer Mannheim. Chloramphenicot was obtained from Sankyo. 12-O-Tetradecanoylphorbol 13-acetate (TPA) was obtained from Dr P. Borchert (Eden Prairie, Minn.). Phorbol was a gift from Drs M. Terada and T. Sugimura. Polyethylene glycol 6000 was purchased from J. T. Baker Chemical (Phillipsburg, N.J.). Hexamethylenebisacetamide (HMBA) was a gift from Dr T. Yamane. Rhodamine 123 was obtained from Eastman Kodak. Eagle's minimum essential medium (MEM, in powder) was purchased from Nissui Seiyaku (Tokyo). Fetal calf serum (FCS) and calf serum were obtained from Flow Lab and Nippon Biotest, respectively. All the reagents used were reagent grade. Exp Cell Res 159 (1985)
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Cells A Friend (murine erythroleukemia) cell line (DS 19) was obtained from Dr M. Terada. This cell line was derived from cell line 745A. A mutant of DS 19, designated as DS 19 (Oua~Cm s) in this paper, lacks functional thymidine kinase (Tk-) and was a gift from Dr R. A. Rifkind. We will not refer to the Tk- mutation in this paper, A double mutant of DS 19 that lacks thymidine kinase and is resistant to ouabain (3 raM), designated as IAM I01 (Oua"CmS), was isolated in this laboratory from N-methylN'-nitro-N-nitrosoguanidine-treated DS 19 (OuaSCm s) cells. These mutant cells are all chloramphenicol-sensitive (Cm~). A chloramphenicol-resistant Friend cells (Cm r, IAM 111) was constructed by introducing Cm r marker into Friend cells (DS 19) from a chloramphenicol-resistant mouse L cell line (B 82 Cm ~) by cytoplast fusion, followed by successive selections starting from low level to relatively high level chloramphenicol resistance (100 ~g/ml). The chloramphenicol-resistant mouse L cell line (13 82 Cm r) [8] was supplied by Dr T. Sekiguchi. The chloramphenicol-resistant Friend cells (IAM 111) exhibited a normal pattern of erythroid differentiation after exposure to DMSO, HMBA and other erythroidinducing agents in the presence or absence of chloramphenicol (80 ~tg/ml) (data not shown).
UV Irradiation Confluently grown cells (2x 106 cells/ml) were collected by centrifugation (500 g, 5 rain) at room temperature and resuspended in phosphate-buffered saline (PBS) at a final cell density of 5 X 1 0 6 cells/ml. The celt suspension (2 ml) was transferred to a plastic Petri dish (60x 12 mm) and irradiated under a Toshiba GL 15 (15 W) germicidal UV lamps at a distance of 52 cm, which gave UV intensity 1.52 J/mZ/sec. Under this condition, approx. 50 % (between 40-60 %) of the ceils survived when we examined their plating efficiency on methylcellulose-containing medium. After irradiation, the cells were collected by centrifugation and resuspended (8x 105 cells/ml) in MEM supplemented with FCS (12 %) for further incubation (24 h) at 37°C.
Preparation of Cytoplasts L cells (B 82 Cm ~) were enucleated according to the method of Wigler et al. [9], in which the cells were centrifuged through Ficoll density gradient in the presence of cytochalasin B. Friend cells were enucleated by the method of Ohara et al. [10]. This method consistently yielded approx. 40% of enucleation and gave a purity of the cytoplast over 99.9 %.
Cytoplast Fusion and Cybrid Selection Cytoplast fusion was performed according to a modification of Pontecorvo's procedure for cell fusion [1 l]. In essence, intact Friend cells (1 x 106 cells) and cytoplasts (3 x 10 6 cells) were first mixed and centrifuged (1 200 g, 5 min) at room temperature. The pellet was mixed with 0.2 ml of polyethylene glycol 6000 (50%, w/w, in water) and kept at room temperature for 2 rain. MEM (1 ml) was then added and, after gentle mixing, the sample was left at room temperature for 3 rain. For cybrid selection, the cells were then diluted with 4 ml of MEM supplemented with calf serum (10%), centrifuged (500 g, 5 rain), and resuspended in 1 ml of MEM containing FCS (12 %), chloramphenicol (80 Ixg/ml) and ouabain (3 raM). After 4 days incubation at 37°C in an incubator with 5 % CO2, oligomycin was added to the selection medium at a final concentration of 10 ng/ml and the cells were incubated for one more day.
Fluorescent Staining Rhodamine 123 staining of the cells was carried out as described by Johnson et al. [12]. Microscopy and photography were performed with a Olympus (BH2-RFK) fluorescence microscope. All photographs were taken using Fujicolor HR 400 film.
Cell Fixation on Polylysine-Coated Coverglass Two or three drops of polylysine solution (1 mg/ml) were placed on a coverglass. After 5 min, the covergtass was washed twice with double-distilled water and dried at room temperature. Cell
Exp CellRes i59 (1985)
Erythroid differentiation in Friend cells 227
Fig. 1. Selection of cybrids between intact cells and rhodamine .t23-1abelled cytoplasts. ][AM 111
(OuaSCm~) cells were labelled with rhodamine 123. Cytoplasts prepared from these cells were fused with IAM 101 (OuarCmS). After 4 days' incubation with chloramphenicol (80 Ixg/ml) and ouabain (3 Exp Cell Res 159 (1985)
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suspension (one or two drops) was placed on the polylysine-coated coverglass and incubated for 2 h at 37°C in an incubator with 5 % COz. After removing the medium and dead cells by aspiration, the coverglasses were rinsed with fresh medium.
RESULTS
Rapid Selection of Cybrids by Sensitization of Non-Fused Cells with Oligomycin In order to characterize the intracellular erythroid inducing activity by cytoplast fusion, we first explored conditions for cybrid selection. In early experiments, cytoplasts (enucleated cells), which were prepared from chloramphenicolresistant (Cm ~) Friend cells (IAM 111), were fused with intact cells (IAM 101) with a nuclear genetic marker (ouabain resistance, Oua r) and the samples were incubated in a selection medium containing chloramphenicol (80 ~tg/ml) and ouabain (3 mM/ml). While a small portion (1%) of the cells which were presumably originated from fused cells (cybrids) between the intact cells (OuarCm s) and the cytoplasts (OuaSCmr) continued to grow in the selection medium, the majority of the cells which consisted of non-fused cells did not lose their viability after 5 days' incubation in the selection medium (data not shown). Since in vitro erythroid differentiation in Friend cells is generally completed within 5 days, it was obvious that selection of cybrids by chloramphenicol and ouabain alone was inadequate for analysing the role of cytoplasmic elements in erythroid differentiation. We suspected that the sustained resistance to chloramphenicol of the nonfused cells was due to residual mitochondrial proteins which are still able to support minimum mitochondrial functions. Accordingly, we employed oligomycin, an inhibitor for oxidative phosphorylation, and found that it greatly accelerated killing of the non-fused cells. For example, when oligomycin (10 ng/ml) was added to the selection medium after 4 days incubation with chloramphenicol (80 ~tg/ml) and ouabain (3 mM), the majority of the cells were killed within 24 h. As seen in table 1, while all the cells remained trypan blue-negative when the cells were incubated with chloramphenicol (and ouabain) alone, addition of oligomycin after 4 days' incubation with chloramphenicol and ouabain converted essentially all the cells to trypan blue-positive cells which consisted of more than 99.9 % of non-fused cells. On the other hand, there was a small but significant difference in the number of trypan blue-negative cells between the fused cells with Cm s cytoplasts and Cm r cytoplasts, indicating selection of cybrids with Cm r cytoplasraM), oligomycin (10 ng/ml) was added to the medium and the cells were incubated for one more day. The cells were then fixed on coverslips coated with polylysine and photographs were taken through a fluorescent microscope (Olympus BH2-RFK). (A) Without; (B) with oligomycin. In order to show a large number of rhodamine-positive cells, the oligomycin-treated samples (B) were concentrated (approx, 100-fold by centrifugation) before applying them to polylysine-coated coverglass slips. The dead cells are not fixed on the slips under this condition. For details, see Materials and Methods. Exp Cell Res 159 (1985)
..... ~- grythroid differentiation in Friend cells 229 Table 1. Cybrid selection by oligomycin Treatment Cytoplast fusion
Cells
Cytoplasts prepared from
IAM 101 (OuarCm s) x IAM 101 (OuarCmS)× DS 19 (Oua~Cm ~) IAM101 (OuarCm~)× I A M I l l (OuaSCm ~)
- Oligomycin
+ Oligomycin
TB ÷
TB-
TB +
TB-
0 0 0
- 1 x 104 - 1 x 104 ~1×104
- 1 x 104 - 1 × 104 ~1×104
3 3 110
Friend cells (IAM 101 OuarCm s) were fused with cytoplasts prepared from either IAM 101 (Oua~Cm s) or IAM 111 (OuaSCm r) as described in Materials and Methods. After 4 days' incubation with chloramphenicol (80 ~tg/ml) and ouabain (3 mM), the samples were further incubated for 1 day with or without oligomycin (10 ng/ml) in addition to chloramphenicol and ouabain. The cells were then stained with trypan blue [0.01% (w/w)]. Trypan blue positive (TB ÷) and negative (TB-) cells (total approx. 104 cells) were counted under a microscope (Olympus BH2-RFK). For details, see Materials and Methods.
mic marker (but not with Cm s marker) under this condition (table 1). From the number of the trypan blue-negative cells appeared from the cell fusion with Cm r cytoplasts, we estimated that approx. 1% of the original cells formed cybrids as a result of fusion with the cytoplasts. The effect of oligomycin on cybrid selection was further confirmed by cell fusion experiments with cytoplasts marked by rhodamine 123 which specifically stains mitochondria. Cytoplasts were first stained with rhodamine 123 and, after their fusion and selection in both the absence and presence of oligomycin in addition to chloramphenicol and ouabain, the samples were examined under a
Table 2. Selection o f cybrids between intact cells and rhodamine 123-labelled cytoplasts--a quantitative analysis
Source of cytoplasts -
DS 19 (Oua~Cm s) IAM 111 (Oua~Cm r)
- Oligomycin
+ Oligomycin
Rho ÷
Rho +
Rho-
Rho-
0
~ 2 × 104
0
0
140 146
~ 2 × 104 ~ 2 x 104
0 101
0 9
DS 19 (Oua~Cm s) and IAM 111 (OuaSCm ~) cells were labelled with rhodamine 123 by the method of Johnson et al. [12]. Cytoplasts prepared from these cells were fused with IAM 101 (OuarCmS). After cybrid selection in the presence or absence of oligomycin (10 ng/ml) as described in Materials and Methods, the cells were fixed on coverglass slips coated with polylysine. Numbers of rhodaminepositive (Rho ÷) and -negative (Rho-) cells (the initial number of the cells; approx. 2x 104 cells) were counted under a fluorescence microscope (Olympus BH2-RFK). The drastic reduction in R h o - cells in oligomycin-treated sample was due to the loss of dead cells (non-fused cells) while washing the samples on polylysine-coated coverglass slips. For details, see Materials and Methods. Exp Cell Res 159 (1985)
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Watanabe, Nomura and Oishi
Table 3. Effect of various compounds on the induction of erythroid-inducing
activity in cytoplasts B ÷ cells (%) Treatment (donor)
Recipient cells - UV + UV
None
0.4
2.5
DMSO HMBA DMSO+TPA DMSO+Phorbol DMSO+Cycloheximide Control (without cell fusion)
1.2 0 0.8 1.0 0.4 0
8.5 6.9 1.4 6.4 0.9 0.4
Friend cells, IAM 111 (Oua~Cmr), were grown as described in Materials and Methods. The cells were then diluted (1 : 1) with a fresh medium and treated with various compounds. After 6 h, the cells were enucleated, fused with IAM 101 (OuarCm S) cells which had been irradiated with UV light (20 J/m 2) 24 h earlier. After cytoplast fusion, the cells were incubated in the presence of chloramphenicol (80 ~tg/ml) and ouabain (3 mM) for 4 days. Oligomycin (10 ng/ml) was then added to the medium and the cells were incubated for one more day. Benzidine-positive (B ÷) cells were then scored. Total of approx. 3x 103 cells were scored for each sample. Concentrations of the compounds used; DMSO 1.8% (v/v), HMBA 5 mM, TPA 100 ng/ml, phorbol 100 ng/ml and cycloheximide 10 ~tg/ml. For the control (without cell fusion) experiment, the cells (IAM I01), with or without UV irradiation, were incubated in the medium (without chloramphenicol and oligomycin) for 5 days and benzidine-positive (B ÷) cells were scored.
f l u o r e s c e n c e m i c r o s c o p e . A typical p i c t u r e o f the c y b r i d s o b t a i n e d with rhodamine 123-stained c y t o p l a s t s ( w i t h o u t o l i g o m y c i n ) is s h o w n in fig. 1 A. As seen in fig. 1 B, addition o f o l i g o m y c i n in the selection m e d i u m essentially eliminated all the n o n - s t a i n e d cells w h i c h c o n s i s t e d o f n o n - f u s e d F r i e n d cells. In table 2, we s h o w the results o f a quantitative analysis o f the e x p e r i m e n t s . It is quite clear that r h o d a m i n e 123-positive cells s u r v i v e d in the selection m e d i u m o n l y w h e n the cell fusion was p e r f o r m e d with C m r c y t o p l a s t s . F u r t h e r m o r e , the e x p e r i m e n t s indicate that the surviving cells in the p r e s e n c e o f c h l o r a m p h e n i c o l (and ouabain) and o l i g o m y c i n were, in fact, derived f r o m the c y b r i d s f o r m e d b e t w e e n intact cells and c y t o p l a s t s .
Characterization of lntracellular Erythroid-Inducing Activity Produced by Erythroid-Inducing Agents B y e m p l o y i n g this rapid c y b r i d selection, we c h a r a c t e r i z e d the intracellular activity i n d u c e d b y typical e r y t h r o i d - i n d u c i n g agents. T h e cells ( I A M 111 OuaSCm r) were i n c u b a t e d with D M S O ( 1 . 8 % , v/v) and, at different time intervals, c y t o p l a s t s were p r e p a r e d f r o m the cells and f u s e d with the cells ( I A M 101 O u a r C m s) w h i c h h a d b e e n irradiated with U V light 24 h before. T h e cells were then s u b j e c t e d to the c y b r i d selection, 4 d a y s ' i n c u b a t i o n with c h l o r a m p h e n i c o l Exp Cell Res 159 (1985)
Erythroid differentiation in Friend cells
,oI
231
10
.
5
~. 0
--3
~
,~...=iff
i
I
i
i
i
!
t
L
i
2
4
6
8
10
2
4
6
8
Time, hr
I
10
Time, hr
Fig. 2. Induction of intracellular erythroid-inducing activity in cytoplasts by DMSO. Friend cells, IAM 111 (OuaSCmr), were grown in MEM supplemented with FCS (12 %) at 37°C to a cell density of 2x106 cells/ml. The cells were then diluted (1 : 1) with the same medium and DMSO was added to a final concentration of 1.8 % (v/v). At different time intervals, the cells were enucleated and cytoplasts were fused with IAM 101 (OuarCm s) cells which had been irradiated by UV light (20 J/m 2) 24 h before. After 4 days incubation with chloramphenicol (80 ~tg/ml) and ouabain (3 raM), oligomycin (10 ng/ml) was added to the medium and the cells were incubated for one more day. Benzidine-positive (B ÷) cells were then scored. Cytoplasts from DMSO-treated cells; fused with O, UV irradiated cells; I , control (-UV) cells. Cytoplasts from control cells; fused with ©, UV-irradiated cells; [], control (-UV) cells. A total of approx. 3 x 103 cells were scored for each sample. For details, see Materials and Methods. Fig. 3. Absence of erythroid-inducing activity in cytoplasts directly treated with DMSO. Friend cells, IAM 111 (OuaSCm~), were grown in MEM supplemented with FCS (12 %) at 37°C to a cell density of 2x 106 cells/ml. The cells were then enucleated. The cytoplasts were suspended in the same medium (1 x l06 cytoplasts/ml) and exposed to 1.8 % (v/v) DMSO for various length of periods. At different intervals as shown in the figure, the cytoplasts were withdrawn, washed once with MEM and mixed with IAM 101 cells (OuarCm s, 1 × 106 cells) which had been irradiated by UV light (20 J/m 2) at 24 h before. After cytoplast fusion, the samples were incubated with chloramphenicol (80 ~tg/ml) and ouabain (3 mM) for 4 days. Oligomycin (10 ng/ml) was then added to the medium and the cells were incubated for one more day. Benzidine-positive (B ÷) cells were then scored. A total of approx. 3 x 103 cells were scored for each sample. For details, see Materials and Methods.
(80 ~tg/ml) and ouabain (3 mM), followed by 1 day incubation with oligomycin (10 ng/ml) in addition to chloramphenicol and ouabain. Cells with accumulated hemoglobin, a characteristic of erythroid cells, were then scored among surviving cells by benzidine staining [13]. As seen in fig. 2, the cytoplasts prepared from DMSO-treated cells were able to induce erythroid differentiation upon fusion with UV-irradiated cells. The extent of the induction (8.5-10% in seven independent experiments) was somewhat lower than that by cell fusion between intact cells, in which 15-20% of the cells became benzidine-positive. As shown in fig. 2, the induction of the activity was not seen upon fusion with control (non-UV-irradiated) cells. These results are consistent with the results of the previous cell fusion experiments that erythroid differentiation in Friend cells is a synergistic result of two distinctive intracellular reactions [6, 7]. Furthermore these results suggest that the intracellular activity Exp Cell Res 159 (1985)
232
Watanabe, Nomura and Oishi
induced by inducing agents exists in cytoplasts, acts in trans and triggers erythroid differentiation as a dominant factor. As also shown in fig. 2, the induction of the activity exhibited a transient nature, although DMSO was continuously present in the medium until the time of preparation of the cytoplasts. The activity in the cytoplasts reached the maximum at 6 h after addition of the inducing agent (DMSO) and declined thereafter. A similar transient nature of the activity had been demonstrated in the cell fusion between two intact Friend cells [7]. The nature of the DMSO-induced activity in cytoplasts was further investigated by examining the effect of biologically active phorbol esters and cycloheximide. We had previously shown that the induction of the erythroid-inducing activity by DMSO (or HMBA) is inhibited by biologically active phorbol esters or cycloheximide. Friend cells (IAM 111 QuaSCmr) were incubated with DMSO together with a typical biologically active phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), or cycloheximide. Cytoplasts prepared from the treated cells were then fused with UV-irradiated or non-irradiated cells and benzidinepositive cells were scored after the cybrid selection. As seen in table 3, it is quite clear that the induction of the activity in the cytoplasts was inhibited by TPA and cycloheximide. Phorbol, a derivative of TPA without a tumor-promoting activity had little effect. We also show that HMBA, another potent erythroid inducer, induced the activity as DMSO did. These results are essentially the same as obtained with previous cell fusion experiments employing intact cells [6, 7], indicating that the erythroid-inducing activity induced in the cytoplasts is the same as that induced in intact cells. As described above, the induction of the erythroid-inducing activity in cytoplasts is inhibited by cycloheximide. This strongly suggests that a protein(s) of nuclear origin (vs mitochondrial origin) is involved in the induction of the activity in the cytoplasts, although the activity remains in the cytoplasts. Probably a signal produced by DMSO or HMBA through a TPA-sensitive trans-membrane reaction reaches to the nuclei at first place. A gene(s) is then activated in responding to the signal and its product(s) is transferred to cytoplasts to express its erythroid-inducing activity. Alternatively, induction of a protein of nuclear origin may be required for the activation of an erythroid-inducing factor which is constitutively present in cytoplasts. In order to further confirm the involvement of nuclei in inducing the activity, we conducted an experiment in which isolated cytoplasts were directly incubated with DMSO and then fused with UV-irradiated cells. As seen in fig. 3, the erythroid-inducing activity was not developed when cytoplasts were directly treated with DMSO, confirming that nuclei play a role in producing the erythroid-inducing activity in cytoplasts. The lack of induction of the activity in the isolated cytoplasts was not due to loss of fundamental physiological functions in the cytoplasts during incubation with DMSO, for the cytoplasts thus treated still maintained at least 80 % of capacity to rescue chloramphenicol sensitive cells after cytoplast fusion (data not shown). Exp Cell Res 159 (1985)
Erythroid differentiation in Friend cells
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DISCUSSION Use of a new rapid selection procedure for cybrids has enabled us to conduct a series of experiments in which erythroid differentiation can be induced by cell (cybrid) fusion with cytoplasts prepared from the cells treated with typical erythroid inducing agents, such as DMSO or HMBA. We showed that cytoplasts thus prepared still maintain an activity which is able to trigger erythroid differentiation upon fusion with UV-irradiated cells. These results indicate that the induced activity, whatever its molecular nature is, is located in cytoplasts and one can trap it in isolated cytoplasts. Furthermore, it has become clear that the activity, as a dominant factor, triggers erythroid induction by a trans-acting manner, provided that the recipient cells had been irradiated by UV light. We further showed that, although the erythroid-inducing activity is located in cytoplasts, the induction is inhibited by cycloheximide. Obviously, nuclei play a role in producing the activity probably through synthesis of a specific protein(s). What is the molecular basis for the activity in the cytoplasts? From the experiments described above alone, it is difficult to determine whether the induced activity resides in cytoplasmic membrane or cytoplasm. If the former is the case, Friend cells may be differentiated into erythroid cells by even partial alteration of their membrane structure caused by the integration of the modified membrane derived from the DMSO (or HMBA)-treated cells. On the other hand, if a cytoplasmic factor is responsible for the activity, the activity may correspond to a defined molecule(s), whose identification, of course, remains to be a subject of future studies. In any event, the activity induced by erythroid-inducing agents acts in trans and seems to be dominant over the pre-existing activity which keeps the cells from committing to erythroid differentiation. Consistent with the previous observations [7], we have shown that cycloheximide blocks the induction of the activity in the cytoplasts, indicating that a protein of nuclear origin is required in inducing the activity. Apparently, a signal produced by a trans-membrane reaction reaches nuclei at first place and then alters expression of a specific gene(s). Probably either the gene product itself corresponds to the activity or it indirectly affects the pre-existing activity in the cytoplasts. Recently, we have identified a novel nuclear protein (p54) in Friend cells whose synthesis is induced by inhibitors for erythroid differentiation, such as biologically active phorbol esters [14]. Constitutive as well as induced synthesis of p54 is inhibited by erythroid inducing agents including DMSO, HMBA and actinomycin D. These results suggest that p54 plays a role in erythroid differentiation in Friend cells as a negatively controlling element. A protein like p54 may be a repressor for the expression of this putative nuclear gene which is responsible for the induction of the erythroid-inducing activity in cytoplasts. There are several reports concerning the role of trans-acting cellular factors in erythroid differentiation [15-18]. Most of such works were carried out with hybrid cells produced by cell fusion between erythroid cells and non-erythroid Exp Cell Res 159 (1985)
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cells. T h e s e r e p o r t s i n d i c a t e that e r y t h r o i d - s p e c i f i c g e n e s are a c t i v a t e d in the h y b r i d cells s u g g e s t i n g the p r e s e n c e o f a trans-acting factor(s) in the d i f f e r e n t i a t e d ceils. A m o n g t h e m , R N A p o l y m e r a s e II was i m p l i c a t e d as o n e o f c r u c i a l c e l l u l a r factors for s u c h a n a c t i v a t i o n [17]o A t the p r e s e n t m o m e n t , the r e l a t i o n s h i p b e t w e e n t h e s e trans-acting c y t o p l a s m i c f a c t o r s a n d the p u t a t i v e f a c t o r d i s c u s s e d in the p a p e r is n o t c l e a r a n d m u s t wait for f u r t h e r i n v e s t i g a t i o n . We are grateful to Ms MJyuki Harada for her assistance in preparing the manuscript. We thank Dr Toyozo Sekiguchi for kindly providing us a chloramphenicol-resistant mouse L cell line used for constructing the Friend cell (IAM 111) and for his valuable suggestions during the course of the study. We also thank Dr Taihei Nozawa for his help in taking pictures of cybrids. This work was supported by Grant-in-Aid for special project research, Cancer-Bioscience, from the Ministry of Education, Science and Culture of Japan.
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Exp CellRes [59 (1985)
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