Proc. Natl. Acad. Sci. USA Vol. 92, pp. 2869-2873, March 1995 Cell Biology

Erythropoietin-induced cellular differentiation requires prolongation of the G1 phase of the cell cycle (/ /erythropoiesis/ 3) MARTIN CARROLL*t, YUAN ZHU*, AND ALAN D. D'ANDREA*t *Division of Pediatric Oncology and Division of Cellular and Molecular Biology, Dana-Farber Cancer Institute, Boston, MA 02115; and tDivision of Hematology and Oncology, Beth Israel Hospital, Harvard Medical School, Boston, MA 02115 Communicated by Gerald D. Fasman, Brandeis University, Waltham, MA, December 12, 1994 (received for review October 26, 1994)

ABSTRACT Erythropoietin (EPO), like many other he- T-cell line, CTLL-EPO-R, with EPO does not result in ery- matopoietic growth factors, can induce either growth or throid differentiation (10). The CTLL-EPO-R cells lack the differentiation of hematopoietic cells. Little is known about requisite erythroid transcription determinants, such as the the molecular basis of this cellular decision, in part because erythroid specific transcription factors GATA-1, NF-E2, and of a paucity of cell lines in which these two phenomena can be EKLF, which complement the EPO-R differentiation re- dissociated. Ectopic expression of the EPO receptor (EPO-R) sponse (10). in Ba/F3, a murine interleukin 3 (IL-3)-dependent progenitor According to the stochastic model, cellular commitment to cell line, confers EPO-dependent cell growth. In these cells differentiation is a random event. In this model, EPO plays a (Ba/F3-EPO-R), EPO also induces 18-globin mRNA, a specific role in erythroid cell viability; the committed erythroid cell has marker of erythroid differentiation. Here we show that the the preprogrammed capacity to become a . EPO induction oferythroid differentiation by EPO requires a delay merely enables the erythroblast to complete this program by in cell growth and a prolongation of the (G1) phase of the cell keeping the cell alive. Evidence for a stochastic model comes cycle. Interestingly, this effect on G1 prolongation was con- from studies of the cellular oncoprotein, Bcl-2 (11). Bcl-2 centration dependent. At low EPO concentrations (0.05-0.1 overexpression in an interleukin 3 (IL-3)-dependent cell line unit of EPO per ml; 1 pM EPO = 0.01 unit of EPO per ml), results in a delay in apoptotic cell death following IL-3 EPO prolonged G1 and induced differentiation; at high con- deprivation. Interestingly, this delay in cell death promotes centrations (0.5-10.0 units per ml), EPO shortened G1 and erythroid differentiation even in the absence of EPO or other preferentially stimulated growth. IL-3 stimulated Ba/F3 growth factors. growth but not differentiation at all growth factor concentra- Recent studies have shown that the cellular decision to tions ranging from 0.1 to 500 pM. Moreover, IL-3 suppressed proliferate or differentiate is strongly affected by the cell cycle. EPO-induced .-globin induction in a dose-dependent manner. In particular, the duration of the G1 phase of the cell cycle This suppression correlated with the shortening of G1 by IL-3. correlates with the probability that a cell will differentiate. Taken together, these data demonstrate distinct effects of Johnson et al. (12) expressed a temperature-sensitive p53 EPO and IL-3 and a balance between erythroid growth and polypeptide in a murine erythroleukemia cell line. At the differentiation that is cell cycle dependent. permissive temperature, murine erythroleukemia cells accu- mulated in G1 and demonstrated increased erythroid differ- Erythropoietin (EPO) is a 34-kDa glycoprotein that is the entiation. In accordance with these results, overexpression of major regulator of mammalian erythropoiesis. EPO circulates cyclin D2 shortened the G1 phase of hematopoietic cells and in the blood and binds to a specific cell surface EPO receptor decreased the probability of differentiation (13). (EPO-R) that is expressed on immature erythroid progenitor EPO may therefore exert its differentiation activity by cells in the bone marrow (1, 2). Paradoxically, EPO has been regulating the cell cycle of responsive erythroid progenitor shown to stimulate either cell growth or cell differentiation, cells. To test this hypothesis, we analyzed the duration of the depending on the specific cell lines or primary cells examined. G1 phase of preerythroid cells growing in different concen- For instance, the murine EPO-dependent cell line, HCD57 (3), trations of EPO or IL-3. Ba/F3-EPO-R cells, which have the proliferates in EPO but does not terminally differentiate. In capacity to grow in either EPO or IL-3, were tested for contrast, primary murine erythroid progenitors differentiate erythroid proliferation or differentiation under these condi- in EPO, thereby upregulating f3-globin mRNA transcription tions. The induction of erythroid differentiation directly cor- and becoming mature erythrocytes (4-6). How a single growth related with the length of the G1 phase. The greater the factor can control either growth or differentiation remains an concentration of EPO used, the greater the initial growth unresolved question. response, the shorter the G1 phase, and the lower the proba- Two plausible models exist for the role of EPO in erythroid bility of differentiation. In contrast, IL-3 never induced 3-glo- differentiation (7, 8). According to the instructive model, EPO bin expression, even at low concentrations (0.01 pM) that drives an uncommitted cell into the erythroid lineage by resulted in a delayed G1 phase. When the two were activating unique biochemical signal transduction pathways. combined, IL-3 suppressed the EPO-induced expression of The best evidence for an instructive model comes from studies ,B-globin mRNA in a dose-dependent manner. These findings of growth factor-dependent cell lines that ectopically express support the concept that there are two discrete signaling the EPO-R. Stimulation of Ba/F3-EPO-R cells with EPO pathways controlled by EPO and IL-3, a differentiative path- drives ,B-globin mRNA accumulation and partial erythroid way and a proliferative pathway. Balance between these path- differentiation (9, 10). Moreover, expression of the EPO-R is necessary but not Abbreviations: EPO, erythropoietin; IL-3, interleukin 3; EPO-R, EPO sufficient for erythroid differentiation. Stimulation of the receptor; FCS, fetal calf serum; MTT, 3-(4,5-dimethyl-2-thiazolyl)- 2,5-diphenyl-2H-tetrazolium bromide; CO, coproporphyrinogen oxi- dase. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed at: Division of payment. This article must therefore be hereby marked "advertisement" in Pediatric Oncology, Dana-Farber Cancer Institute, 44 Binney Street, accordance with 18 U.S.C. §1734 solely to indicate this fact. Boston, MA 02115. 2869 Downloaded by guest on September 29, 2021 2870 Cell Biology: Carroll et al. Proc. Natl. Acad Sci. USA 92 (1995) ways can be altered by modulating the concentration of these Cell Cycle Analysis. Fluorescence intensity in each cell cytokines. nucleus was measured with a FACScan (Becton Dickinson). Starved or growth-factor-stimulated cells (106) were fixed in 40% ethanol for 30 min and stained with propidium iodide. MATERIALS AND METHODS Approximately 10,000 cells were analyzed at a rate of 50-100 Cells and Transfection. Ba/F3 cells (14) were maintained in cells per second by the FACScan. The percentage of cells in complete RPMI 1640 medium containing 1 pLM 2-mercapto- each phase of the cell cycle was determined by analyzing data ethanol, 10% heat-inactivated fetal calf serum (FCS), and 5% with the computer program CELLFIT (Becton Dickinson). Data (vol/vol) RPMI 1640 medium conditioned by WEHI cells as a shown are representative of at least three separate growth source of murine IL-3. Ba/F3-EPO-R cells (clone 22; ref. 10) factor induction experiments. were grown in complete RPMI 1640 medium containing 10% FCS and supplemented with recombinant murine IL-3 (Kirin RESULTS Brewery, Tokyo) (10 pM) and G418 (1 mg/ml). For induction, cells were washed in RPMI 1640/10% FCS and resuspended To clarify the relationship between EPO-stimulated cellular in the same medium containing recombinant human EPO differentiation and growth, we analyzed Ba/F3-EPO-R cells (Genetics Institute, Cambridge, MA) at the indicated concen- growing in either IL-3 or EPO (Fig. 1). As shown in Fig. 1A trations. Cells were cultured in an atmosphere of 5% C02/ and consistent with previous reports (9, 10), Ba/F3-EPO-R 95% air at 37°C. For transfections, Ba/F3 cells were trans- cells (subclone 22) grown in EPO (0.05 unit per ml) expressed fected with the plasmid, pLXSN-EPO-R by electroporation as ,B-globin mRNA, while cells grown in IL-3 (1 pM) did not. A described (10) and selected in RPMI 1640/10% FCS/5% kinetic analysis of cell growth at these growth factor concen- G418 (1 mg/ml). trations demonstrated an initial growth lag in EPO at day 2, WEHI-conditioned medium containing compared to growth in IL-3 (Fig. 1B). After 3 days in EPO, DNA and RNA. The pLXSN-EPO-R construct was derived growth increased, suggesting that the initial lag was not simply by inserting the EPO-R cDNA into the EcoRI restriction a poor mitogenic stimulus. To further characterize the EPO- enzyme site of pLXSN. Total RNA was isolated from the induced delay in cell growth, we next performed cell cycle indicated cell lines by the guanidium isothiocyanate/cesium analysis (Fig. 1C). Cells were synchronized in early G1 by chloride method. Northern blot analysis was carried out ac- growth factor deprivation and then stimulated with either IL-3 cording to the supplier's recommendations: 15 ,ug of total or EPO. As shown in Fig. 1C, growth factor deprivation RNA was fractionated through a 1% agarose/formaldehyde resulted in the arrest of 75-80% of the cell population in Go gel, transferred to a nylon membrane, and hybridized with a or G1. Cells stimulated with EPO entered and remained in the random-primed labeled cDNA probe at 68°C for 2 h in G1 phase for greater than 24 h. Although flow cytometric QuikHyb buffer (Stratagene). Blots were washed at high analysis did not distinguish Go and G1 populations, most cells stringency (68°C; 0.1% SSC; lx SSC = 0.15 M NaCl/0.015 M entered G1 phase within 1 h of being stimulated, as judged by sodium citrate) and exposed to Kodak XAR film. their accumulation of c-myc mRNA (16) (data not shown). Cell Proliferation Assay. Individual subclones expressing Cells stimulated with IL-3 exited G1 and entered S phase the EPO-R were assayed for growth in RPMI 1640 medium/ within 10 to 15 h. Erythroid differentiation in the presence of 10% FCS supplemented with several concentrations of recom- EPO, therefore, correlated with prolongation of the G1 phase. binant human EPO or recombinant murine IL-3. Cell growth To determine the significance of G1 prolongation, we next and viability were measured by using the trypan blue exclusion measured 0-globin mRNA accumulation and cell growth assay (14) and the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl- parameters at multiple concentrations of EPO (Fig. 2). EPO 2H-tetrazolium bromide (MTT) assay (Sigma) as described induced dose-dependent growth of Ba/F3-EPO-R cells (Fig. (15). Each cell-growth experiment was performed three times 2A), as described (14). The concentration of EPO that induced with similar results each time. For dose-response studies, cells half-maximal growth was 0.5 unit per ml. Interestingly, there were plated in 96-well plates at a density of 2 x 104 cells per was an inverse relationship between EPO-induced /3-globin ml (200 ,ul) and grown for 72 h. The effect of each IL-3 or EPO mRNA accumulation and EPO concentration between 0.05 concentration on cell proliferation was assayed in triplicate. and 0.5 unit per ml (Fig. 2A Inset). This concentration range A B lo'- C 1loo _ - 28S 70 - 60 - *-o * CO = 706t) - *1' - 18S u <-,lot, 0 u e- 40- vs 30 - .c. V' P-GLOBIN -_ 0 2( - 10 -

_ 0 5 10 15 20 25 30 1 2 Time, days Time after stimulation, h

FIG. 1. Erythropoietin induces 13-globin mRNA accumulation by prolongation of the G, phase of the cell cycle. (A) Total RNA was isolated from Ba/F3-EPO-R subclone 22 after 72 h of growth in either 1 pM murine IL-3 (lane 1) or 0.05 unit of EPO per ml (1 pM EPO = 0.01 unit of EPO per ml; lane 2). Total RNA (15 jig per lane) was analyzed by electrophoresis, Northern blotting, and hybridization with a 32P-labeled murine f3-globin cDNA probe. Equal sample loading was confirmed by rehybridizing the blot with a cDNA probe to the constitutively expressed human glyceraldehyde-3-dehydrogenase (data not shown). (B) Cellular growth in 0.05 unit of EPO per ml (-) or 1 pM IL-3 (O) for the indicated times was measured by the trypan blue exclusion assay (14). (C) Washed Ba/F3-EPO-R cells were deprived of growth factor for 12 h and then stimulated with 0.05 unit of EPO per ml (-,A) or 1 pM IL-3 (O, V) for the indicated times. Cells were subsequently stained with propidium iodide and subjected to FACS analysis, as previously described. The percentage of cells in the Go and G, phases (-, O) or in the S phase (A, V) was calculated from DNA flow cytometry histograms and plotted as shown. Downloaded by guest on September 29, 2021 Cell Biology: Caffoll et aL Proc. NatL Acad Sci. USA 92 (1995) 2871

C 100 11B~~ 907 .05 .1 .5 80, EPO, unit per ml 1.0 70 u 60 0 0 50 4-i / ~~~u ( 40 0.5 30 6 & z

0.1 1 10 0 2.5 5 5 10 15 20 25 30 EPO, unit per ml Time, days Time after stimulation, h

FIG. 2. Inverse relationship between EPO-induced ,B-globin mRNA accumulation and EPO concentration. (A) EPO-dependent growth characteristics of Ba/F3-EPO-R subclone 22 cells. Cells were washed three times in RPMI 1640 medium and resuspended in RPMI 1640 supplemented with 10% FCS and various concentrations of recombinant human EPO. Cell growth and viability were measured by the MTT assay (Sigma) at 450 nm (15). (Inset) Northern blot of )3-globin mRNA induced at the indicated EPO concentration. The position of 13-globin mRNA is indicated by an arrow. (B) Cell growth in EPO at a concentration of either 0.5 (-), 0.1 (*), or 0.05 (-) unit of EPO per ml. (C) Percentage of cells in the Go and G1 phases. EPO concentrations and symbols are the same as in B. Shown for comparison is the percentage of Go/G1 cells grown in 1 pM IL-3 from Fig. 1 (O).

corresponded to the physiological range of EPO-induced concentration of IL-3 was similar to that observed with EPO growth. A similar inverse relationship has previously been at 0.5 unit per ml. However, at a low concentration of IL-3 described in EPO-responsive murine erythroleukemia cells (0.01 pM), cells did not express f3-globin mRNA (Fig. 3A, lane (12). At both a low (0.05 unit per ml) and an intermediate (0.1 2), irrespective of a growth delay (Fig. 3B) and a prolonged G1 unit per ml) concentration of EPO, there was a significant lag phase (Fig. 3C). Thus, the EPO-R promoted erythroid dif- in cell growth (Fig. 2B) and a prolongation of G, (Fig. 2C) ferentiation at a low EPO concentration and mitogenesis at a compared to growth in 1 pM IL-3. At a high concentration of high EPO concentration, while the IL-3 receptor was incapable EPO (0.5 unit per ml) there was no significant lag in cell of promoting a differentiation response at any tested concen- growth and no concomitant prolongation of G1. tration. These data highlight important functional differences These results suggested that prolongation of G, was neces- between the EPO-R and the IL-3 receptor. Furthermore, they sary for ,B-globin mRNA induction. To determine if G1 demonstrate that GI prolongation is necessary but not suffi- prolongation was sufficient for induction, we next analyzed cell cient for f3-globin mRNA induction. growth and 3-globin mRNA induction over a wide range of To determine which growth factor effect was dominant, we IL-3 concentrations (Fig. 3). Murine recombinant IL-3 in- examined combinations of EPO and IL-3 on erythroid differ- duced dose-dependent growth of Ba/F3-EPO-R cells (Fig. entiation and cellular growth (Fig. 4). At a constant concen- 3A). The concentration of IL-3 that induced half-maximal tration of EPO, sufficient for ,B-globin mRNA induction (0.05 growth was 1 pM, consistent with previous studies using unit per ml), IL-3 induced a dose-dependent suppression of IL-3-dependent cell lines (17). At 1 pM IL-3, cells did not erythroid differentiation (Fig. 4A). Suppressing concentra- express 13-globin mRNA (Fig. 3A, lane 4), did not demonstrate tions of IL-3 consistently abrogated EPO-induced growth a growth delay (Fig. 3B), and were not maintained in a delay (Fig. 4B) and stimulated rapid entry of cells into S phase protracted G1 (Fig. 3 C). Response to this comparatively high (Fig. 4C). This suggested that in the presence of a mitogenic

__

0.05 .01 0.1 1.0 EPO. IL-3, pM B 10(- A 1.5~unit/ml C

80 v) 70 1 - -d 60 - \ U4 C 50 - 40 - 0.5 e 30 -

20 -

1 -

o l l 0 - I l 0 2.5 5 7.5 10 0.001 0.01 0.1 1.0 l1 100 1000 5 10 15 20 25 30 IL-3, pM Time, days Time after stimulation, h

FIG. 3. G1 prolongation is necessary but not sufficient for induction of f-globin mRNA accumulation. (A) IL-3-dependent growth characteristics of Ba/F3-EPO-R subclone 22 cells. Cells were washed three times in RPMI 1640 medium and resuspended in RPMI 1640 supplemented with 10% FCS and various concentrations of recombinant murine IL-3. Cell growth and viability were measured as described in the legend to Fig. 2. (Inset) Northern blot analysis of ,3-globin mRNA marked by arrow induced by the indicated IL-3 or EPO concentration. (B) Cell growth in 0.5 unit of 1 ~~~~-0 EPO per ml (-) or IL-3 at pM (O), 0.01 pM (El), or 0.001 pM (0). (C) Percentage of cells in the Go and G1 phases after stimulation with 1 pM (K) or 0.01 pM (o) IL-3. Shown for comparison is the percentage of Go/61 cells following growth in 0.05 unit of EPO per ml (0).

Downloaded by guest on September 29, 2021 ,- 2872 Cell Biology: Carroll et al. Proc. Natl. Acad Sci USA 92 (1995)

A IL-3. pM 10 - 1.0 0.01 0.001 B log v C 100 EPO, unit/mli - .05 .05 .05 .05 90 80- 5-GLOBIN -01 510*7-U .. 70 0- U, / o~~~~~~~~~60 1 2 3 4 5 j3o6- ..50- 0 30 4 iDs1 20- 10 ~ 1 104 I 0 0 2.5 5 7.5 10 0 5 10 15 20 25 30 Time, days Time after stimulation, h

FIG. 4. IL-3 suppresses EPO-induced 13-globin mRNA accumulation. (A) Northern blot analysis of ,B-globin mRNA induced by EPO (0.05 unit per ml) in the absence or presence of various concentrations of murine IL-3. (B) Cell growth in either EPO alone (0.05 unit per ml) (0) or EPO (0.05 unit per ml) plus 1 pM (V), 0.01 pM (El), or 0.001 pM (0) IL-3. (C) Percentage of cells in the Go and G1 phases observed for cells grown in either EPO (0.05 unit per ml) alone (-), IL-3 (1 pM) alone (K), or EPO plus IL-3. Symbols are the same as in B. stimulus the G1 phase is shortened and erythroid differenti- DISCUSSION previous ation is suppressed. Consistent with these results, In the current study, we used the Ba/F3-EPO-R cell system to studies have demonstrated that IL-3 suppresses granulocyte investigate the early events required for EPO-induced ery- colony-stimulating factor-induced myeloid differentiation of throid differentiation. In particular, we have studied the 32D cells (18, 19). relationship between cell growth and differentiation. Growth A low concentration of IL-3 partially suppressed 13-globin in low concentrations of EPO resulted in an initial delay in cell mRNA induction by EPO (Fig. 4A, lane 4). Cells grown in 0.01 growth, protraction of G1, and subsequent accumulation of pM IL-3 and 0.05 unit of EPO per ml demonstrated a delay in ,B-globin mRNA. Conversely, high concentrations of EPO cell growth (Fig. 4B) and G1 prolongation (Fig. 4C). Taken induced a strong mitogenic signal, shortened G1, and thereby together, these results suggest that the suppressive activity of blocked erythroid differentiation. This demonstrates that EPO IL-3 on erythroid differentiation may not be solely related to can signal growth or differentiation in a particular cell line its growth stimulatory effects. depending on its concentration. In contrast, the multilineage To ensure that accumulation of ,B-globin mRNA repre- growth factor IL-3 did not induce differentiation at any dose, sented a bona fide differentiation event, we next tested EPO- confirming the distinct effects of these two cytokines. Also, for induced Ba/F3-EPO-R cells for evidence of other erythroid- this model system, IL-3 effectively suppressed erythroid dif- specific markers. Coproporphyrinogen oxidase (CO; EC ferentiation in a dose-dependent manner. 1.3.3.3) is an erythroid-specific enzyme involved in heme Low concentrations of EPO (0.05 unit per ml) induced metabolism (20). The mRNA for CO has been shown to ,B-globin mRNA as early as 12 h after growth factor addition accumulate during erythroid differentiation (21). We analyzed (data not shown). At this EPO concentration, 80% of the three independent subclones of Ba/F3-EPO-R for their ability Ba/F3-EPO-R cells remain in G1 for up to 30 h (Fig. 1C). to undergo erythroid differentiation. In response to low con- Taken together, these results suggest that j-globin mRNA centrations of EPO (0.05 unit per ml) each subclone induced induction occurs in the absence of DNA replication and cell f3-globin mRNA and CO mRNA (Fig. 5). None of the sub- division. Alternatively, a small percentage of Ba/F3-EPO-R cells that exit G1 at this EPO concentration may account for clones synthesized hemoglobin (data not shown). The Ba/F3- the large ,B-globin mRNA signal. The biochemical mechanism EPO-R cells, therefore, provide a useful model system for of /3-globin mRNA accumulation following the EPO stimulus studying partial but not complete erythroid differentiation. is not known. Accumulation could result from increased transcription, decreased mRNA degradation, or preferred *-CO mRNA cytoplasmic transport and processing of globin-specific nu- clear transcripts (22). IL-3 fails to induce erythroid differentiation, even at low concentrations that allow G1 prolongation. Furthermore, IL-3 suppresses EPO-induced j3-globin mRNA accumulation. The 411:..:... mRNA ability of IL-3 to suppress differentiation may result from the nature of the particular cell system used. For instance, in contrast to our results, previous studies using bone marrow cell 1 2 3 4 5 6 cultures have suggested that IL-3 potentiates EPO-induced

I Il__1 L._J erythroid burst formation (23). Immortalized, IL-3-dependent subclone subclone subclone cell lines, such -as Ba/F3, have been selected for continuous 1 2 3 growth and absence of differentiation. The IL-3 receptor may therefore specifically activate a signal transduction mechanism FIG. 5. EPO induces partial erythroid differentiation of Ba/F3- that suppresses erythroid differentiation in these cells. EPO-R subclones. Ba/F3 cells were transfected with pLXSN-EPO-R Our data support both the stochastic model and the instruc- and selected in G418 and IL-3. Three subclones of Ba/F3-EPO-R cells tive model of erythroid differentiation. Consistent with the were isolated by limiting dilution. The subclones (105 cells) were stochastic model, Ba/F3-EPO-R cells have the inherent ability washed and resuspended in medium containing either IL-3 (1 pM) or to differentiate into erythroid cells. These cells express the EPO (0.5 unit per ml) for 72 h. Total RNA was analyzed by Northern factors blotting by using a 32P-labeled full-length cDNA for murine f3-globin prerequisite erythroid-specific transcription including or murine CO (21). Lanes 1, 3, and 5, RNA samples from cells GATA-1, NF-E2, and EKLF and are therefore primed for stimulated with IL-3; lanes 2, 4, and 6, RNA samples from cells differentiation. Low concentrations of EPO enable G1 pro- stimulated with EPO. longation and erythroid differentiation. Consistent with the Downloaded by guest on September 29, 2021 Cell Biology: Carroll et aL Proc. NatL Acad Sci USA 92 (1995) 2873 instructive model, the growth signal induced by EPO is 6. Koury, M. J., Bondurant, M. C. & Mueller, T. J. (1986) J. Cell. qualitatively different from the signal by IL-3. Low concen- Physiol. 126, 259-265. trations of either EPO or IL-3 result in G1 prolongation; 7. Nakahata, T., Gros, A. J. & Ogawa, M. (1982) J. Cell. Physiol. however, only EPO exposure results in the of 113, 455-458. induction 8. Till, J. E., McCullogh, E. A. & Siminovitch, L. (1964) Proc. Natl. erythroid differentiation. The EPO-R and the IL-3 receptor Acad. Sci. USA 51, 29-36. activate distinct combinations of JAK kinases and STAT 9. Chiba, T., Nagata, Y., Kishi, A., Sakamaki, K, Miyajima, A., transcription factors (29). It is likely that these biochemical Yamamoto, M., Engel, J. D. & Todokoro, K (1993) Proc. Natl. differences account for the qualitative differences in cellular Acad. Sci. USA 90, 11593-11597. outcome. 10. Liboi, E., Carroll, M., D'Andrea, A. D. & Mathey-Prevot, B. Finally, EPO-induced G1 prolongation may account, at least (1993) Proc. Natl. Acad. Sci. USA 90, 11351-11355. in part, for various discrepant results reported in the literature. 11. Fairbairn, L. J., Cowling, G. J., Reipert, B. M & Dexter, T. M. (1993) Cell 74, 823-832. First, several studies have suggested that EPO provides pri- 12. Johnson, P., Chung, S. & Benchimol, S. (1993) Mol. Cell. Biol. 13, marily either a mitogenic (24) or a differentiative (25) signal. 1456-1463. Our studies suggest that different EPO concentrations may 13. Kato, J.-Y. & Sherr, C. J. (1993) Proc. Natl. Acad. Sci. USA 90, have accounted for these different cellular outcomes. Second, 11513-11517. recent conflicting studies using chimeric cytokine receptors 14. D'Andrea, A. D., Yoshimura, A., Youssoufian, H., Zon, L. I., identified a putative differentiation domain in either the Koo, J.-W. & Lodish, H. F. (1991) Mol. Cell. Biol. 11, 1980-1981. extracytoplasmic region (26) or the cytoplasmic region (27, 28) 15. Mosmann, T. (1983) J. Immunol. Methods 65, 55-63. of the EPO-R polypeptide. Again, these discrepant findings 16. Kelly, K, Cochran, B. H., Stiles, C. D. & Leder, P. (1983) Cell 35, 603-610. may have resulted from differences in the EPO concentration 17. Holmes, K L., Palaszynski, E., Fredrickson, T. N., Morse, H. C., used for induction or in the nature of the specific chimeric III & Ihle, J. N. (1985) Proc. Natl. Acad. Sci. USA 82,6687-6691. utilized. The chimeric cytokine receptor 18. Ziegler, A. F., Bird, T. A., Morella, K K, Mosley, B., Gearing, polypeptides may differ in their inherent abilities to influence D. P. & Baumann, H. (1993) Mol. Cell. Biol. 13, 2384-2390. the length of the G1 phase of the cell cycle. For these previous 19. Fukunaga, R., Ishizaka-Ikeda, E. & Nagata, S. (1993) Cell 74, studies, the variable effects of EPO and EPO-R on- cellular 1079-1087. differentiation should be reinterpreted in terms of EPO con- 20. Grandchamp, B., Beaumont, C., de Verneuil, H. & Nordmann, centration and concomitant cell cycle changes. Y. (1985) J. BioL Chem. 260, 9630-9635. 21. Kohno, H., Furukawa, T., Yoshinaga, T., Tokunaga, R. & Taketani, S. (1993) J. Biol. Chem. 268, 21359-21363. We thank C. Corless for technical assistance. We are indebted to our 22. Bondurant, M. C., Lind, R. N., Doury, M. J. & Ferguson, M. E. colleagues B. Mathey-Prevot, M. Pless, D. G. Nathan, C. Sieff, S. Mer- (1985) Mol. Cell. BioL. 5, 675-683. chav, and D. Barber for critical comments and for a careful reading of 23. Sieff, C. A., Niemeyer, C. M., Nathan, D. G., Ekern, S. C., the manuscript. This work was supported by a grant from the National Bieber, F. R., Yang, Y. C., Wong, G. & Clark, S. C. (1987)J. Clin. Institutes of Health (ROl DK43889-03), from the Sandoz Corporation Invest. 80, 818-823. (A.D.D.), from the American Cancer Society (M.C.), and from the 24. Koury, M. J. & Bondurant, M. C. (1990) Science 248, 378-381. Leukemia Society of America (Y.Z.). A.D.D. is a Lucille P. Markey 25. Imada, K, Tsudo, M., Kodaka, T.-i., Itoh, K, Arima, N., Hattori, Scholar, and this work was also supported in part by a grant from the T., Okuma, M. & Uchiyama, T. (1992) Biochem. Biophys. Res. Lucille P. Markey Charitable Trust. Commun. 188, 352-357. 26. 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