Differentiation of Erythroid Progenitor (CFU-E) Cells from Mouse Fetal ...

Vol. 8, No. 6

MOLECULAR AND CELLULAR BIOLOGY, June 1988, p. 2604-2609 0270-7306/88/062604-06$02.00/0 Copyright © 1988, American Society for Microbiology

Differentiation of Erythroid Progenitor (CFU-E) Cells from Mouse Fetal Liver Cells and Murine Erythroleukemia (TSA8) Cells without Proliferation TSUYOSHI NOGUCHI, HIROAKI FUKUMOTO, YUJI MISHINA, AND MASUO OBINATAt* Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan Received 3 September 1987/Accepted 2 March 1988

Erythropoietin (epo) appears to play a significant role in influencing the proliferation and differentiation of erythroid progenitor (CFU-E) cells. To determine the mechanism of action of epo, the effect of drugs on the in vitro colony formation of CFU-E cells induced from a novel murine erythroleukemia cell line, TSA8, was examined. While cytosine arabinoside inhibited colony formation and terminal differentiation of the CFU-E cells responding to epo, herbimycin, which is a drug that inhibits src-related phosphorylation, inhibited colony formation only. The same effect of herbimycin was observed with normal CFU-E cells from mouse fetal liver cells. These results suggest that epo induces two signals, one for proliferation and the other for differentiation, and that the two signals are not linked in erythroid progenitor cells. been shown to be a selective inhibitor of the phosphorylation of p60`rc protein in cells, through screening of agents that are active in converting the transformed morphology of Rous sarcoma virus-infected normal rat kidney cells to a normal morphology (23, 24). In this work, we demonstrated that herbimycin inhibited the action of epo on the proliferative response, but not on the differentiation response, of erythroid progenitor cells.

Mature hematopoietic cells, such as erythrocytes and granulocytes, are derived from several types of progenitor cells, each committed to develop into only one or two cell lineages, but all progenitor cells originate from the same stem cells. Thus, the maintenance of hematopoiesis requires a balance between self-renewal and differentiation (2, 6, 22). The progenitor cells, immature progeny of the stem cells, are recognized by their ability to produce colonies in vitro in the presence of specific growth-regulating molecules (5, 22). Erythropoietin (epo) is a well-characterized molecule and induces the in vitro formation of erythroid progenitor (CFUE) cell colonies synthesizing hemoglobins in a semisolid medium (2, 5, 12, 21). The differentiating colony is formed as a result of the proliferation and differentiation of progenitor cells induced by epo action. Apparently, the growth-regulating molecules require two signals, one for cell proliferation and the other for differentiation. Previously, we demonstrated that a novel murine erythroleukemia cell line, TSA8, can be induced to undergo commitment to differentiation into CFU-E-like progenitor cells with several inducers in vitro (15). The properties of the CFU-E colonies of induced TSA8 cells are very similar to those of CFU-E colonies from fetal liver cells in the following respects: (i) they consist of more than eight cells, (ii) they are hemoglobin positive, and (iii) they are epo dependent (15, 17). We found that TSA8 cells possess surface epo receptors before induction, but some factor(s) in addition to epo receptors is required to acquire responsiveness to epo action (17). This in vitro system provides a suitable model with which to examine how proliferation and differentiation are induced by epo action in erythroid precursor cells. To know more about the responsiveness to epo of these CFUE-like progenitor cells induced from TSA8, we surveyed inducers other than dimethyl sulfoxide (DMSO) and drugs which affected the differentiation or proliferation responses to epo of the CFU-E-like progenitor cells. We found that herbimycin elicited an interesting response. Herbimycin has

MATERIALS AND METHODS Culture and induction of commitment of TSA8 cells. TSA8 cells were established by Shibuya and Mak (20) from anemia-inducing Friend virus complex, FV-A. The cell line was kindly provided by T. W. Mak of the University of Toronto, Toronto, Canada. The properties of the cell line have been described previously (15). The induction protocol for TSA8 cells is essentially similar to that described earlier (18). The cells were grown in Iscove modified Dulbecco medium supplemented with 15% fetal bovine serum, as described elsewhere (8). For induction, the inducer was added to a cell suspension at a density of 2 x 105 cells per ml. For better induction, slightly overgrown cells were passaged and the inducer was added 1 day after passage, just after the cells had started to grow. The CFU-E assay was carried out in methylcellulose by using the techniques of Iscove et al. (8). The cells were transferred to a semisolid medium at various times after the addition of the inducer. Cells (4 x 103) were plated in a 24-well multidilution dish (Coming Glass Works, Coming, N.Y.) in a mixture containing Iscove modified Dulbecco medium, 0.8% methylcellulose (Tokyo Chemical Industries, Tokyo, Japan), 1% bovine serum albumin (Filtoron, Inc.), 100 mM ,-mercaptoethanol, and 0.5 U of step III sheep plasma epo (Connaught, Canada) per ml. Dishes were incubated at 37°C in a humidified atmosphere flushed with 5% CO2 in air. Colonies were directly stained with benzidine on day 2 and scored with an inverted microscope. To examine the effect of herbimycin on the formation of CFU-E-like colonies, colonies or single cells stained with benzidine were differentially scored. Herbimycin was obtained from Y. Uehara, National Institute of Health, Tokyo, Japan. Preparation of fetal erythroid precursor cells. Pregnant ICR

* Corresponding author. t Present address: Department of Cell Biology, The Research Institute for Tuberculosis and Cancer, Tohoku University, Seiryo-

machi 4-1, Sendai 980, Japan. 2604

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TABLE 1. Effect of herbimycin on induction of commitment of TSA8 cells Herbimycin

Growth inhibition

% Colonies

(ng/ml)

iton

formed in semisolid

0 100 200 300 500

100 24 23 15 15

37.0 ± 3.1 15.4 ± 0.25 5.9 ± 0.67 0 0

concn

mediuma

%

Benzidinepositive colonies' 47.1 ± 5.0 51.1 ± 5.3 43.6 ± 3.0

a Values (means + standard deviations) are percentages of the total number of colonies formed from 4,000 cells seeded into the semisolid medium. b Values (means ± standard deviations) are percentages of the number of benzidine-positive colonies relative to the total number of colonies.

mice were sacrificed on day 12 of pregnancy, and fetal livers were removed. The livers were separated into single cells with a 26-gauge needle in a medium (GIBCO Laboratories, Grand Island, N.Y.) and washed twice with the same medium. These cells contained approximately 5% CFU-E cells (14). To remove mature erythrocytes, we used the lysing procedure described by Boyle (1), with a slight modification (15). Fetal liver cells were suspended in a solution containing 155 mM NH4Cl, 10 mM KHCO3, and 1 mM EDTA, left for 15 min at 4°C to allow the destruction of mature cells, and then washed once with the same buffer. The cells were then used for the CFU-E assay as described above. Cell cycle analysis of TSA8 cells with a cell sorter. TSA8 cells were induced with DMSO, and herbimycin was added to one culture. Cell growth was monitored by cell count for 2 days. At various times, the cells were fixed with ethanol and digested with pancreatic ribonuclease. Cells were stained with propidium iodide or with ethidium bromide and analyzed by cell sorters in the laboratories of K. Okumura at Juntendo Medical School and of H. Sato at Tokyo Medical and Dental University. RESULTS Effect of herbimycin on the induction of commitment of TSA8 cells to differentiation into CFU-E-like progenitor cells. Initially, the effect of herbimycin on the induction of commitment of TSA8 cells to differentiation into CFU-E-like progenitor cells with DMSO was examined (Table 1). Herbimycin at various concentrations was added with DMSO (1%) at day 0 to liquid cultures of TSA8 cells. After 2 days of culture, the cell number was counted and the inhibition of cell growth was calculated. Without the drug, the cell density was 2.3 x 106 cells per ml. The cells were washed once with phosphate-buffered saline to remove herbimycin, and 4,000 cells were seeded into one well in semisolid medium containing epo (0.5 U/ml) and methylcellulose (8%). After 2 days in semisolid medium, the colonies formed were stained with benzidine, and then the number of epo-dependent differentiated colonies (CFU-E-like colonies) formed was scored. When herbimycin was added to the liquid culture of TSA8 cells with DMSO, cell growth was inhibited, depending on the drug concentration. The exposure of TSA8 cells in the liquid culture inhibited colony formation even in the semisolid medium without herbimycin. Colony formation was completely inhibited at 300 ng/ml. On the other hand, the proportion of epo-dependent benzidine-positive colonies did not change with the addition of herbimycin. Thus, herbimycin had no apparent inhibitory effect on the commitment of

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TSA8 cells to differentiation into CFU-E-like progenitor cells at the concentration which inhibited both cell growth and colony formation. Also, herbimycin inhibited the growth of TSA8 cells, but not the induction of commitment with DMSO. The number of epo-dependent, benzidine-positive single cells increased when that of colonies formed decreased because of the pretreatment of TSA8 cells with increasing concentrations of herbimycin in the liquid culture (Table 1). The proportion of benzidine-positive single cells to total single cells is essentially similar to that of benzidinepositive colonies to total colonies (data not shown). Thus, the commitment of TSA8 cells to differentiation into CFUE-like progenitor cells occurs without proliferation, and once the cells are committed, the differentiation occurs dependent on epo and without proliferation. This finding indicates the separation of the proliferation and differentiation responses to the effects of herbimycin, although at a stage sometime earlier than the CFU-E-like stage. Inhibition by herbimycin of proliferation response, but not of differentiation response, of CFU-E-like progenitor cells to epo. The effect of herbimycin on the CFU-E-like progenitor cells induced from TSA8 cells was then examined. Herbimycin was added to the induced TSA8 cells after the cells were transferred to the semisolid medium. The complete inhibition of colony formation was observed at a lower concentration (30 ng/ml) (Fig. 1). The inhibitory effect of herbimycin

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conc of herbimycin (ng/mI) FIG. 1. Effect of herbimycin on formation of colonies by CFUE-like progenitor cells induced from TSA8 cells and by CFU-E cells from mouse fetal liver cells. TSA8 cells were induced with DMSO for 2 days in a liquid culture, and 2 x 103 cells were screened in the semisolid medium, at different concentrations of herbimycin. After 2 days of incubation in the presence of epo, the cells were stained with benzidine and the numbers of three types of colonies were scored. The fetal liver cells of 13-day-old mice were treated by the lysing procedure described in Materials and Methods and seeded in the semisolid medium, at different concentrations of herbimycin. After 2 days of incubation with epo, the colonies were stained with benzidine and the positive colonies were scored. Symbols: 0, nondifferentiating colonies; O, partly stained colonies; *, CFU-Elike colonies; 0, CFU-E colonies from mouse fetal liver cells.

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FIG. 2. Differentiated cells in semisolid medium observed in the presence of herbimycin. TSA8 cells were induced with DMSO in a liquid culture, seeded in the semisolid medium in the absence (A) or presence (B) of herbimycin (30 ng/ml), and cultured for 2 days. The cells were stained with benzidine. Positively stained single cells (single arrow), two positively stained cells together (two arrows), and two cells together, of which one cell is positively stained (half stained; three arrows), are shown.

the formation of three types of colonies, nondifferentiating, partly stained, and CFU-E-like colonies, was essentially the same (Fig. 1); thus, this inhibition is not due only to the transformed phenotype. Herbimycin inhibited the formation of nondifferentiating colonies. The nondifferentiating colonies of TSA8 cells in the semisolid medium may resemble an anchorage-independent tumor cell growth, which is one of the specific properties of transformed cells. We then examined the effect of herbimycin on the formation of CFU-E colonies obtained from normal tissues. CFU-E cells were obtained from mouse fetal liver cells, as described earlier (14). The effect of herbimycin on the formation of normal CFU-E colonies in the presence of epo was similar to, although slightly weaker than, that observed in the CFU-E-like progenitor cells induced from TSA8 cells (Fig. 1). Thus, herbimycin also inhibits the colony formation of normal erythroid progenitor cells induced with epo. To determine the mechanism of the inhibitory effect of herbimycin on the formation of CFU-E colonies, cells in the semisolid culture were carefully examined. At a herbimycin concentration of 30 ng/ml, although colony formation was strongly inhibited, single cells or two divided cells were usually observed. When cells were stained with benzidine, we detected three classes of cells: positively stained single or two divided cells; two neighboring cells, one positive and the other negative; and nonstained cells (Fig. 2). The positively stained cells were only present in the semisolid culture with on

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Then, we examined the dose-dependent effect of herbimycin on the formation of CFU-E-like colonies and positively stained single cells. The number of CFU-E-like colonies decreased, and that of positively stained single cells increased, depending on the concentration of herbimycin (Fig.

3, top). Without herbimycin, there were 250 benzidinepositive colonies and 20 benzidine-positive single cells. At a herbimycin concentration between 30 and 50 ng/ml, no benzidine-positive colonies were detected, but approximately 200 benzidine-positive single cells were present. The total number of cells seen as colonies or as single cells gradually decreased but did not change significantly in response to drug concentration. Thus, the possibility can be ruled out that herbimycin may simply interfere with the ability of induced cells to remain adherent to each other and thereby prevent colony formation. It is clear that the proportion of differentiated cells was not altered by the presence of herbimycin and that only the formation of colonies was inhibited by the drug. Since the positively stained single cells appeared only with the addition of epo in the presence of herbimycin, it is likely that epo can induce the differentiation phenotype of the CFU-E-like progenitor cells derived from TSA8 cells without cell proliferation. We examined whether normal CFU-E cells can be differentiated by epo without cell proliferation. To do this, CFU-E cells were partially purified by a lysing procedure which destroyed mature erythroid cells (1). Thereafter, erythroid cells from fetal liver cells were seeded in the semisolid medium with or without epo in the presence of different concentrations of herbimycin. Figure 3 (bottom) shows the effect of herbimycin on the appearance of the CFU-E colonies and the hemoglobin-positive single cells, depending on the epo action. While the number of CFU-E colonies decreased when the concentration of herbimycin was increased, that of positively stained single cells increased. Thus, herbimycin inhibited the proliferation of CFU-E cells but did not inhibit their differentiation. This finding strongly

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indicates that epo induces differentiation of normal erythroid progenitor cells without cell division. We then wanted to determine whether a similar inhibitory effect occurs with a known inhibitor of DNA synthesis (Fig. 4). In contrast to herbimycin, cytosine arabinoside inhibited both the proliferation and differentiation of CFU-E cells from mouse fetal liver cells and of CFU-E-like progenitor cells. Thus, the inhibition of DNA synthesis may affect the differentiation program induced by the epo action. Effect of herbimycin on cell cycle of TSA8 cells. To explain the selective action of herbimycin on the commitment of TSA8 cells to differentiation into CFU-E-like progenitor cells and on the formation of differentiated colonies of CFU-E by epo, we investigated the cell cycle at which this inhibitor blocks the cells. TSA8 cells were induced in a liquid culture with DMSO, and herbimycin (300 ng/ml) was added to one culture. Herbimycin began to arrest the increase of cell number at 3 h after addition, and the cell number

FIG. 3. Effect of herbimycin on colony formation of induced TSA8 cells or CFU-E cells from mouse fetal liver cells. (Top) TSA8 cells were induced with DMSO for 2 days in a liquid culture, and 2 x 103 cells were seeded in the semisolid medium, at different concentrations of herbimycin. After 2 days of incubation in the presence of epo, the cells were stained with benzidine and the number of positive colonies or the number of positively stained single cells was scored. The number of positively stained single cells included the number of two positively stained cells together. In a separate experiment, 2 x 103 cells were seeded in the semisolid medium without epo and the number of positive colonies was scored. (Bottom) The fetal liver cells of 13-day-old mice were treated by the lysing procedure described in Materials and Methods and seeded in the semisolid medium, at different concentrations of herbimycin. After 2 days of incubation with or without epo, the cells were stained with benzidine and the positive colonies and positively stained single cells were scored. Symbols: 0, positively stained colonies in the presence of epo; *, positively stained single cells in the presence of epo; 0, positively stained colonies in the absence of positively stained single cells in the absence of epo; V, cells epo; seen as colonies or as single cells. l,

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NOGUCHI ET AL.

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remained constant for 48 h of treatment (Fig. 5A). At various times after drug addition, the cells were taken from the culture, fixed, and stained. The stained cells were analyzed with a cell sorter. After drug addition, the cells in the S phase decreased with time in culture, but those in the Gl phase and in the G2/M boundary showed no apparent change even after 48 h of drug treatment (Fig. 5B); thus, the cells were blocked at certain stages of the Gl phase, and/or the G2/M boundary affected neither the induction of commitment of TSA8 cells to differentiation into CFU-E-like progenitor cells nor the differentiation of CFU-E cells by epo.

DISCUSSION In the mouse, CFU-E cells appear to represent a stage of pathway-restricted (i.e., committed) erythroid differentiation; epo appears to play a significant role in influencing the proliferation and differentiation of erythroid progenitor cells (2, 5-7, 11, 19, 22). However, little is known of the molecular aspects of the regulation of progenitor cell proliferation by epo. We have recently demonstrated that TSA8 cells can be induced to undergo commitment to differentiate into CFUE-like progenitor cells (15). This in vitro system may be a good one with which to examine the action of epo on erythroid progenitor cells. The effect of herbimycin on the colony formation of CFU-E-like progenitor cells induced from TSA8 cells is quite interesting. Herbimycin inhibited the effects of epo on proliferation but not on induction of differentiation (Fig. 3, top). The same effect was observed with the CFU-E cells from fetal mouse liver cells (Fig. 3, bottom). The effect of herbimycin on the formation of CFU-E colonies (Fig. 6) indicates that two signals, for differentiation and for proliferation, are separately controlled by epo. Waneck et al. (25) reported that Abelson murine leukemia virus-infected cells are epo independent, whereas Harvey murine sarcoma virus-infected cells are epo dependent; Harvey murine sarcoma virus can only initiate colony formation, while Abelson murine leukemia virus can both stimulate growth and drive differentiation. Thus, at least two signals may be required for complete colony maturation, and the interplay between onc gene products and normal signals for growth and differentiation may be important. The result of Waneck and his colleagues is compatible with our observation. Uehara et al. (23, 24) reported that after herbimycin treatment, the rate of intracellular phosphorylation of p6osr'

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FIG. 5. Cell cycle analysis of induced TSA8 cells treated with herbimycin. TSA8 cells were induced with 1.0% DMSO in the presence or absence of herbimycin (300 ng/ml) and collected at various times. The cells were fixed with 70% ethanol and treated with pancreatic ribonuclease (1 ng/ml) for 30 min at 37°C. Then, DNA in the cells was stained with propidium iodide (50 ,ug/ml), and cells were analyzed by a cell sorter. (A to F) Results for cells from 0, 3, 6, 12, 24, and 48 h of culture after the addition of herbimycin; (G) results for cells from a 12-h culture without herbimycin.

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MECHANISM OF ACTION OF ERYTHROPOIETIN

in normal rat kidney cells infected with Rous sarcoma virus drops to 20 to 40% of that of nontreated cells. The difference is mostly the result of a selective decrease in the phosphorylation of p6Osrc itself. Phosphorylation of bulk cellular proteins is less affected. While the selective action of herbimycin on the phosphorylation of p6Osrc is rather clear, its selective effects on the cellular onc gene products have not been demonstrated. However, it is plausible that phosphorylation of a src-related onc gene product is involved in the signal for the proliferative response. The effect of herbimycin on tyrosine-specific phosphorylation of cellular proteins of TSA8 cells was analyzed by using antiphosphotyrosine antiserum. Preliminary results showed that the phosphorylation of 55- and 30-kilodalton species was selectively inhibited after herbimycin treatment (T. Noguchi, unpublished observation). Thus, it seems likely that herbimycin inhibits the proliferation of CFU-E in the presence of epo by inhibiting the phosphorylation of these proteins, although a further investigation of the nature of the tyrosine-phosphorylated proteins involved in erythroid progenitor cells will be necessary. The mechanism of action of a src-related gene product in erythroid progenitor cells (9, 10) may be important in the elucidation of the control of the balance between proliferation and differentiation of the progenitor cells. Commitment has been thought to be limited to a specific phase of the cell cycle (4, 6, 18). Herbimycin-treated TSA8 cells can pass through one S phase but are arrested at the Gi phase and at the G2/M boundary (Fig. 5). The effect of herbimycin suggests that the commitment occurs at the Gi phase and/or at the G2/M boundary but not at the S phase. Our results indicate that cell division is not required for the commitment of CFU-E cells to differentiation by epo action or to the commitment of TSA8 cells to differentiation into CFU-E-like cells. In any case, this in vitro system may be adequate for the investigation of the mechanism involved in the reprogramming of a differentiation-specific gene during commitment.

throid leukemia cell: a stochastic analysis. Cell 9:221-229. 7. Hankins, W. D., J. Schooley, and C. Eastment. 1986. Erythropoietin, an autocrine regulator? Serum-free production of erythropoietin by cloned erythroid cell lines. Blood 68:263-268. 8. Iscove, N. N., F. Siever, and K. H. Witerhalter. 1974. Erythroid colony formation in culture of mouse and human bone marrow: analysis of the requirement for erythropoietin by gel filtration and affinity chromatography on agarose-concanavalin A. J. Cell. Physiol. 83:309-320. 9. Kahn, P., B. Adkins, H. Beug, and T. Graf. 1984. src- and fps-containing avian sarcoma viruses transform chicken erythroid cells. Proc. Natl. Acad. Sci. USA 81:7122-7126. 10. Kahn, P., L. Erykberg, C. Brady, I. Stanley, H. Beug, B. Vennstrom, and T. Graf. 1986. v-erbA cooperates with sarcoma oncogenes in leukemic cell transformation. Cell 45:349-356. 11. Kranz, S. B., and E. Goldwasser. 1984. Specific binding of erythropoietin to spleen cells infected with the anemia strain of Friend virus. Proc. Natl. Acad. Sci. USA 81:7574-7578. 12. Lee-Huang, S. 1984. Cloning and expression of human erythropoietin cDNA in Escherichia coli. Proc. Natl. Acad. Sci. USA 81:2708-2712. 13. McDonald, J. D., F.-K. Lin, and E. Goldwasser. 1986. Cloning, sequencing, and evolutionary analysis of the mouse erythropoietin gene. Mol. Cell. Biol. 6:842-848. 14. Mishina, Y., T. Kato, A. Urabe, F. Takaku, S. Natori, and M. Obinata. 1986. Unique pattern of gene expression in the erythroid precursor cells. Dev. Growth & Differ. 28:1-6. 15. Mishina, Y., and M. Obinata. 1985. Induction of commitment of murine erythroleukemia cell (TSA8) to CFU-E with DMSO. Exp. Cell Res. 162:319-325. 16. Noguchi, T., H. Fukumoto, Y. Mishina, and M. Obinata. 1987. Factors controlling induction of commitment of murine erythroleukemia (TSA8) cells of CFU-E (colony forming unit erythroid). Development 101:169-174. 17. Ogawa, M., P. N. Porter, and T. Nakahata. 1983. Renewal and commitment to differentiation of hematopoietic stem cells (an interpretive review). Blood 61:823-829. 18. Ostertag, W., I. B. Pragneli, B. Fagg, T. M. Jovin, B. G. Grimwade, and D. J. Arndt-Jovin. 1980. Cell cycle dependent events during Friend cell differentiation. In G. B. Rossi (ed.), In vivo and in vitro erythropoiesis: the Friend system. Elsevier Biomedical Press, Amsterdam. 19. Ramirez, F., R. Gambino, G. M. Maniatis, R. A. Rifkind, P. A. Marks, and A. Bank. 1975. Changes in globin mRNA content during erythroid cell differentiation. J. Biol. Chem. 250:60546058. 20. Shibuya, T., and T. W. Mak. 1983. Isolation and induction of erythroleukemia cell lines with properties of erythroid progenitor burst-forming cells (BFU-E) and erythroid precursor cells (CFU-E). Proc. Natl. Acad. Sci. USA 80:3721-3725. 21. Stephenson, J. R., A. A. Axelrad, D. L. McLeod, and M. M. Shreeve. 1971. Induction of colonies of hemoglobin-synthesizing cells by erythropoietin in vitro. Proc. Natl. Acad. Sci. USA 68: 1542-1546. 22. Till, J. E., and E. A. McCulloch. 1980. Hemopoietic stem cell differentiation. Biochim. Biophys. Acta 605:431-459. 23. Uehara, Y., M. Hori, T. Takeuchi, and H. Umezawa. 1985. Screening of agents which convert 'transformed morphology' of Rous sarcoma virus-infected rat kidney cells to 'normal morphology': identification of an active agent as herbimycin and its inhibition of intracellular src kinase. Jpn. J. Cancer Res. (Gann) 76:672-675. 24. Uehara, Y., M. Hori, T. Takeuchi, and H. Umezawa. 1986. Phenotypic change from transformed to normal induced by benzoquinonoid ansamycins accompanies inactivation of p6O.rc in rat kidney cells infected with Rous sarcoma virus. Mol. Cell. Biol. 6:2198-2206. 25. Waneck, G. L., L. Keyes, and N. Rosenberg. 1986. Abelson virus drives the differentiation of Harvey virus-infected erythroid cells. Cell 44:337-344.

ACKNOWLEDGMENTS We thank S. Natori for helpful discussions, K. Okumura and H. Sato for cell cycle analysis, Y. Uehara for supplying herbimycin, and A. R. Frackelton, Jr., for supplying antiphosphotyrosine antiserum. This work was supported by Grants-in-Aid for Cancer Research and for Scientific Research from the Ministry of Education, Science and Culture of Japan. LITERATURE CITED 1. Boyle, W. 1968. An extension of the Cr-release assay for the estimation of mouse cytotoxins. Transplantation 6:761-764. 2. Dexter, T. M., E. Spooncer, R. Schofield, B. 1. Lord, and P. Simmons. 1984. Haemopoietic stem cells and the problem of self-renewal. Blood Cells 10:315-339. 3. Djaldetti, M., H. Preisler, P. A. Marks, and R. A. Rifkind. 1972. Erythropoietin effects on fetal mouse erythroid cells. II. Nucleic acid synthesis and the erythropoietin-sensitive cells. J. Biol. Chem. 247:731-735. 4. Gambari, R., P. A. Marks, and R. A. Riflind. 1979. Murine erythroleukemia cell differentiation: relationship of globin gene expression and of propagation of Gl to inducer effects during Gl/early S. Proc. Natl. Acad. Sci. USA 76:4511-4515. 5. Goldwasser, E. 1984. Erythropoietin and its mode of action. Blood Cells 10:147-162. 6. Gusella, J., R. Geller, B. Clarke, V. Weeks, and D. Houseman. 1976. Commitment to erythroid differentiation by Friend ery-

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