Proc. Natl. Acad. Sci. USAVol. 80, pp. 210-214, January 1983Cell Biology

Indirect induction of erythroid differentiation in mouse Friendcells: Evidence for two intracellular reactions involved inthe differentiation

(cell fusion/DNA damage/tumor promoter)

SHINTARO NOMURA AND MICHIO OISHIInstitute of Applied Microbiology, University of Tokyo, Tokyo, Japan

Communicated by Charlotte Friend, September 30, 1982

ABSTRACT The mechanism of in vitro erythroid differentia-tion in mouse Friend cells was studied by employing cell fusionbetween two genetically marked Friend cells and other nonery-throid cells, including BHK (baby hamster kidney) and FM3A(mouse mammary gland) cells. We were able to induce erythroiddifferentiation indirectly by fusing Friend cells that had been ex-posed briefly to dimethyl sulfoxide prior to fusion with nonery-throid cells that had been treated with ultraviolet light (or otherDNA-damaging agents). The results suggest that two distinct re-actions are involved in erythroid differentiation in Friend cells invitra One reaction, originating from the damaged DNA (or in-hibition of DNA replication as a consequence), exhibits an induc-ible nature, is nonspecific to Friend cells, and is tranm-acting. Theother reaction is specific to Friend cells and most likely is cis-act-ing. We also present evidence from the cell fusion experimentsthat a typical tumor promoter, 12-0-tetradecanoylphorbol 13-ace-tate, inhibits erythroid differentiation by affecting the latter re-action. The biological significance of these findings is discussed.

Friend cells (murine erythroleukemia cells) (1) can be inducedin vitro to express various cellular functions characteristic ofnormal erythropoiesis, including synthesis of hemoglobinmRNA, hemoglobin, and heme, appearance of erythrocytemembrane antigens, and cessation of cell division (2-6). Theinducers for this in vitro erythroid differentiation in Friend cellsare mostly nonphysiological agents such as dimethyl sulfoxide(Me2SO) (2), butyric acid (7, 8), or hexamethylenebisacetamide(9). Besides these potent inducers, a variety ofagents that affectDNA metabolism, such as UV light, x-ray, and bleomycin, in-duce erythroid differentiation of Friend cells (10-13). The in-ducing activities of these agents are generally rather weak, butsometimes they can act synergistically with other nonphysio-logical inducing agents (11, 12).

In order to further investigate the mechanism of the effectsof inducing agents, especially agents that affect DNA metabo-lism, on erythroid differentiation in Friend cells, we undertooka series ofexperiments using cell fusion between two geneticallymarked Friend cell lines and other nonerythroid cells that hadbeen subjected to a variety of inducing conditions. We havesucceeded in inducing erythroid differentiation indirectly byfusing Friend cells with nonerythroid cells that had been treatedwith UV irradiation (or another DNA-damaging treatment),provided that Friend cells had been briefly exposed to Me2SO.These indirect induction experiments suggest that two funda-mentally different mechanisms are involved in erythroid dif-ferentiation of Friend cells. More specifically, both a trans-membrane signal and a signal derived from modified DNAstructure are essential for the in vitro erythroid differentiation.

The signal from DNA seems to involve a trans-acting substance,which is not necessarily specific to Friend cells. The significanceofthis finding in erythroid differentiation in vitro, including thesite of the inhibition of tumor promoter on erythroid differ-entiation, will be also discussed.

MATERIALS AND METHODSMaterials. Thymidine, aminopterin, hypoxanthine, and

methotrexate were obtained from Sigma. Ouabain (g-strophan-thin) was purchased from Boehringer Mannheim. 12-O-Tetra-decanoylphorbol 13-acetate (TPA) and phorbol were generousgifts from M. Terada and T. Sugimura. Polyethylene glycol 6000was obtained from J. T. Baker Chemical (Phillipsburg, NJ).Eagle's minimal essential medium (in powder) was purchasedfrom Nissui Seiyaku (Tokyo). Fetal calfserum was obtained fromFlow Laboratories. Calf serum was supplied by Granite Diag-nostics (Burlington, NC).

Cells. A Friend (murine erythroleukemia) cell line (DS19)was obtained from M. Terada. This cell line was derived fromcell line 745 (2). A mutant of DS19, designated DS19(Tk-) inthis paper, which lacks functional thymidine kinase, was a giftfrom R. A. Rifkind. DS19(Hprt-), a mutant of DS19, presum-ably having a defective hypoxanthine phosphoribosyltransfer-ase, was isolated in this laboratory from N-methyl-N'-nitro-N-nitrosoguanidine-treated DS19 cells as a clone resistant to 6-thioguanine at 15 ,ug/ml. A double mutant of DS19 that lacksthymidine kinase and is resistant to ouabain (1.5 mM), desig-nated DS19(Tk-Ouar), was isolated from N-methyl-N'-nitro-N-nitrosoguanidine-treated DS19(Tk-) cells.A FM3A cell line, originally established from mouse mam-

mary tumor (14), and its thymidine kinase-deficient mutant (Bu-7) were supplied by D. Ayusawa. A doubly marked FM3A,FM3A(Tk-Mtxr), which lacks functional thymidine kinase andcarries methotrexate-resistant dihydrofolate reductase, was ob-tained by transformation of FM3A(Bu-7) with DNA fromChinese hamster ovary (CHO) cells (A29) resistant to metho-trexate at 0.2 ,ug/ml (15). A BHK-21 (baby hamster kidney) cellline was obtained from G. Tamura.

Cell Culture. Friend cells were cultured and maintained inEagle's minimal essential medium supplemented with 12.5%heat-inactivated fetal calf serum. FM3A cells were grown inminimal essential medium supplemented with 12.5% heat-in-activated fetal calf serum. BHK-21 cells were cultured in min-imal essential medium containing 10% heat-inactivated calfserum. The medium used for the selection of the fused cellscontained hypoxanthine (0.1 mM), aminopterin (0.4 AM), thy-midine (16 ,AM), and glycine (3 ,uM) (HAT medium) in minimal

Abbreviations: Me2SO, dimethyl sulfoxide; TPA, 12-0-tetradecanoyl-phorbol 13-acetate; HAT medium, hypoxanthine/aminopterin/thy-midine/glycine medium.

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Proc. Natd Acad. Sci. USA 80 (1983) 211

medium supplemented with 12.5% fetal calfserum. All the cul-tures were incubated in plastic Petri dishes (Falcon, 60 X 15mm) or multiwell plates (Falcon, 24 or 96 wells) at 370C in hu-midified atmosphere containing 5% CO2 in air.UV Irradiation. For Friend and FM3A cells, confluently

grown cells (2 x 106/ml) were collected by centrifugation (1,200x g, 5 min) at room temperature, washed once with phosphate-buffered saline (137mM NaCl/4.2mM KCl/9.6mM Na2HPO4/1.1 mM KH2PO4) and resuspended in phosphate-buffered sa-line at a final cell density of 3-5 x 106 cells per ml. The cellsuspension (0.5 ml) was transferred to a plastic Petri dish (60X 12 mm) and irradiated for various time periods under a To-shiba GL15 (15-W) germicidal UV lamp at a distance of 52 cm,1.35 J/m2-sec. After irradiation, the cells were collected by cen-

trifugation and resuspended in the fetal calf serum/minimalessential medium (4 x 105 cells per ml) for further culture at370C. BHK cells, grown to a density of 1.5-2 x 106 cells perdish (60 x 15 mm), were transferred to fresh minimal essentialmedium supplemented with 10% calfserum (5 ml per dish, 0.3-0.4 X 106 cells per dish). After 2 days, the medium was removedby suction, and the cells were rinsed once with phosphate-buff-ered saline and irradiated.

Cell Fusion and Selection. Cell fusion was performed ac-

cording to a modified procedure of Pontecorvo (16). Cells weregrown to a density of 2 X 106 per ml and 5 x 105 cells of eachline to be fused were mixed and centrifuged (1,200 X g, 5 min)at room temperature. (For BHK cells, the cells were firsttreated with trypsin at 0.5 mg/ml in phosphate-buffered salinebuffer containing 0.02% EDTA for 5 min at 37C and resus-

pended in minimal essential medium containing 10% fetal calfserum at a cell density of 5 x 105 per ml.) After removing thesupernatant, the pellet was mixed with 0.2 ml of polyethyleneglycol 6000 [50% (wt/wt) in water] and kept at room temper-ature for 2 min. Minimal essential medium (1 ml) was thenadded and, after gentle mixing, kept at room temperature for3 min. Fetal calf serum/minimal essential medium (4 ml) wasadded, and, after thorough mixing, the cells were sedimented(600 x g, 5 min) and resuspended in 1 ml of fetal calf serum-supplemented minimal essential medium for further incuba-tion. Sixteen hours later 10 ,ul of 100x HAT solution was addedto the medium and the cells were incubated for 5 days at 370C.When selection was made for methotrexate or ouabain resis-tance as well, methotrexate or ouabain was added to the HATmedium at final concentration of 0.2 ug/ml or 1.5 mM, re-

spectively. When the cells were grown in the medium withmethylcellulose, at 16 hr after cell fusion, the cells were cen-

trifuged, washed once with phosphate-buffered saline, and re-

suspended in HAT medium containing methylcellulose, 1.5%(wt/vol), for further incubation.

Assay of Erythroid Differentiation. Hemoglobin synthesis,characteristic of erythroid differentiation, was assayed accord-ing to the method of Orkin et aL (17) by benzidine staining ofthe hemoglobin accumulated in the cells.

RESULTS AND DISCUSSION

When Friend cells are exposed to 1.8% (vol/vol) Me2SO for acertain period of time (30-35 hr in our culture condition) thecells acquire the capacity to differentiate into erythroid cellseven in the absence ofthe drug thereafter. We have found that,after a shorter period (15-25 hr) of exposure to Me2SO, only asmall portion (2-3%) ofFriend cells were induced into erythroiddifferentiation, but the frequency increased to over 20% whenthe cells had been first irradiated with a moderate dose of UVlight (20 J/m2, which gave 55-70% killing). The erythroid dif-ferentiation caused by this dose ofUV irradiation alone was only

slightly higher than the background value (0.5-4%). It appearedthat UV irradiation and briefexposure to Me2SO exercise a syn-ergistic or concerted action to induce Friend cells to differen-tiate, consistent with previous observations (11, 12). The otheragents that affect DNA structure and DNA metabolism, in-cluding mitomycin C and novobiocin, also exhibited a similareffect on erythroid differentiation when combined with briefMe2SO exposure, but potent erythroid inducers such as butyricacid and N-methylacetamide did not show such a synergisticeffect when combined with a Me2SO pulse (data not shown).Therefore, it seems likely that disturbance ofDNA metabolism,probably inhibition of DNA replication, somehow potentiatesFriend cells to commit to erythroid differentiation triggered bya Me2SO pulse. The reaction triggered by UV irradiation seemsto be a type of induction. The maximal induction of erythroiddifferentiation was observed when UV-irradiated cells were in-cubated more than 10 hr before exposure to Me2SO. The stim-ulatory effect of UV irradiation gradually decreased when in-tervals between UV irradiation and Me2SO exposure wereshortened. Only very low levels of erythroid induction wereobserved when the cells were irradiated at the end of or afterthe Me2SO exposure (data not shown).

In order to explore the nature of the cellular potential in-duced by DNA-damaging agents, the effect of inducing treat-ments on erythroid differentiation was examined by fusing twodifferent genetically marked Friend cells that had been sub-jected to different treatments, DS19(Tk-) with a mutation inthe thymidine kinase (tk) gene and DS19(Hprt-) with a muta-tion in the hypoxanthine phosphoribosyltransferase (hprt) gene.These cells (tk-hprt' and tk'hprt-) themselves will not growin selective medium (HAT medium), but the fused cells withboth tk' and hprt' genes should grow on HAT medium. Whenthese two cells were fused and plated in HAT medium, ap-proximately 1% of the cells originally present gave rise to col-onies. Because no such colonies (less than 0.01%) were observedafter the fusion between cells with identical genetic back-grounds, it is reasonable to assume that the colonies producedin HAT medium were derived from the fused cells with twodifferent genetic backgrounds, complementing each other, tobe able to grow in HAT medium. Table 1 shows the results froma cell fusion experiment, in which DS19(Tk-) and DS19(Hprt-)were treated either by UV light (25 J/m2) or Me2SO (1.8% for16 hr), fused, incubated in selective medium, and assayed for

Table 1. Erythroid differentiation of fused Friend cellsTreatment Benzidine-positive

DS19(Tk-) DS19(Hprt-) cells, %0.1

UV 3.8UV 4.0UV UV 4.5

Me2SO 1.7Me2SO 1.5Me2SO Me2SO 2.6UV Me2SO 33.7

Me2SO UV 35.8

DS19(Tk-) and DS19(Hprt-) Friend cells were grown in fetal calfserum-supplemented minimal essential medium to a cell density of 2x 106 cells per ml. One of the cell preparations (Tk- or Hprt-) wasexposed to 1.8% (vol/vol) Me2SO for 16 hr and the other was irradiatedby UV light (20 J/m2) and incubated for 24 hr. Cell fusion was thenperformed, followed by incubation in fetal calf serum-supplementedminimal essential medium for 20 hr. The cells were then collected,washed once with phosphate-buffered saline, and incubated in fetal.calf serum-supplemented HAT medium. Benzidine-positive cells werescored on the 5th day.

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erythroid differentiation by benzidine staining. As is seen fromthe table, a substantial percentage (33-35%) ofthe cells becamebenzidine positive among cells derived from cell fusion betweenUV-irradiated DS19(Tk-) (or HprtV) and Me2SO-treatedDS19(HprtV) (or TiV). On the other hand, the frequency of theerythroid differentiation with the fused cells between UV-ir-radiated DS19(Tk-) and similarly treated DS19(Hprt-) or be-tween Me2SO-treated DS19(Tk-) and similarly treatedDS19(HprtV) was substantially lower (2-4%), without therebeing any synergistic or even additive effect of the treatments.In similar experiments, when fused cells were plated on solidmedium, colonies consisting of majority of benzidine-positivecells also appeared in the same fashion as in the suspension cul-ture shown in Table 1. This is shown in Fig. 1. Essentially thesame results were also obtained with fusion of mitomycin C-treated (or bleomycin-treated) cells and Me2SO-pulsed cells(data not shown). In preliminary experiments, we observed thata butyric acid pulse could substitute for a Me2SO pulse. Theseresults suggest that the combination ofUV irradiation (or treat-ment by DNA-damaging agents) and Me2SO (or butyric acid)pulse induce two, probably qualitatively distinct, intracellularreactions, which act synergistically to commit Friend cells toerythroid differentiation.Use of the cell fusion described above has made it possible

to further analyze the mechanism of erythroid differentiationby first inducing these intracellular reactions in two physicallyseparated entities and then by reconstituting these reactionsafter cell fusion. We first investigated the specificities of thereactions induced by UV irradiation and Me2SO pulse. For thispurpose, we fused nonerythroid mouse FM3A(Tk-Mtxr) cells,

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FIG. 1. Erythroid differentiation in fused Friend cells as a functionof time after cell fusion. Two genetically marked Friend cells,DS19(Tk-) and DS19(Hprt-), were grown in fetal calf serum-supple-mented minimal essential medium to a cell density of 2 x 10' cells perml. DS19(Tk-) cells were exposed to 1.8% (vol/vol) Me2SO for 16 hr.DS19(Hprt-) cells were irradiated with UV light (20 J/m2) and incu-bated for 24 hr. Control cells (no UV irradiation or Me2SO treatment)were incubated for the same length of time as the treated cells. Cellfusion was then performed, followed by incubation in fetal calf serum-supplemented minimal essential medium. At 16 hr, the cells were col-lected, washed once with phosphate-buffered saline, and incubated infetal calf serum-supplemented HAT medium containing 1.5% (wt/vol) methylcellulose (18). Benzidine-positive (B+) colonies were thenscored every day thereafter as indicated on the abscissa. Colonies thatconsisted of more than 90% benzidine-positive cells were counted asB+ colonies. *, Me2SO-treated DS19(Tk-) fused with UV-irradiatedDS19(Hprt-); o, Me2SO-treated DS19(Tk-) fused with DS19(Hprt-);o, DS19(Tk-) fused with UV-irradiated DS19(Hprt-); *, DS19(Tk-)fused with DS19(Hprt-).

which have defective thymidine kinase and methotrexate-resistant dihydrofolate reductase, with wild-type FriendDS19(Tk+Mtx') cells and examined whether UV or Me2SOtreatment of the FM3A cells can induce erythroid differentia-tion upon fusion with the Friend cells. Fig. 2 shows that UVirradiation of FM3A cells can induce erythroid differentiationafter fusion with Me2SO-pulsed Friend cells. On the otherhand, Me2SO treatment of FM3A cells did not potentiate oraffect the erythroid differentiation ofUV-irradiated Friend cellsupon cell fusion. In order to further confirm this observation,other cell fusions involving different genetic markers were per-formed. These included cell fusion between (i) a wild-typeBHK-21 (Tk+Ouas) cell line, which has functional thymidinekinase (Tk+) and is sensitive to 1.5 mM ouabain (Ouas), and adouble mutant ofFriend cells, DS19(Tk-Ouar), which lacks thy-midine kinase and is resistant to ouabain (1.5 mM) and (ii) awild-type FM3A (Tk+Ouas) line and the double mutant of Friendcells, DS19(TkIOuar), which lacks thymidine kinase and is re-sistant to ouabain. The results, including control experimentswith cell fusion between two Friend cells, are shown in Figs.3, 4, and 5. Consistent with results ofthe previous experiments(Fig. 2), UV irradiation of BHK cells or FM3A cells inducederythroid differentiation after cell fusion with Me2SO-pulsedFriend cells. Again, Me2SO treatment of these nonerythroidcells did not affect erythroid differentiation upon fusion withUV irradiated Friend cells. As seen from these figures, the op-timal UV dose for maximal erythroid induction after fusion withcells exposed to 1.8% Me2SO for 16 hr ranged between 20 and30 J/m2.

It has become quite clear from these experiments that theUV-induced cellular reaction(s) required for erythroid differ-entiation in Friend cells in vitro can be induced not only inFriend cells but also in nonerythroid cells, suggesting a wide

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FIG. 2. Effect of UV irradiation and Me2SO treatment on ery-throid differentiation in fusions between FM3A cells and Friend cells.(A) FM3A(Tk MtXr) cells that had been irradiated with UV light atvarious intensities (shown on the abscissa) and then incubated for 16hr were fused with Me2SO-pulsed [1.8% (vol/vol), 16 hr] (.) or un-treated (o) Friend cells (DS19). After cell fusion, the cells were incu-bated in HAT medium containing methotrexate (0.2 ,ug/ml) for 5 daysand benzidine-positive (B+) cells were scored. (B) Similar to A, butFriend cells (DS19) that had been irradiated with UV at various in-tensities (abscissa) and incubated for 16 hr were fused with Me2SO-pulsed [1.8% (vol/vol), 16 hr] (e) or untreated (o) FM3A(Tk-Mtxr)cells. In other control experiments, fusion between Me2SO-pulsed[1.8% (vol/vol), 16 hr] FM3A(Tk-Mtx) and similarly treated Friendcells (DS19) andbetweenUV-irradiated (25J/m2) FM3A cells and sim-ilarly treated Friend cells gave 2.6% and 3.3% benzidine-positive cells,respectively.

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Proc. Nad Acad. Sci. USA 80 (1983)

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Proc. Natl. Acad. Sci. USA 80 (1983) 213

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FIG. 3. Effect of UV irradiation, and Me2SO treatment on ery-throid differentiation in fusions between BHK-21 cells and Friendcells. (A) BHK-21 cells that had been irradiated with UV light at var-ious intensities (abscissa) and then incubated for 16 hr were fused withMe2SO-pulsed [1.8% (vol/vol), 16 hr] (o),or untreated (o) DS19(TkrOuaDcells. After cell fusion, the cells were incubated in HAT medium con-taining ouabain (1.5 mM) for 5 days and benzidine-positive (B+) cellswere scored. (B) Similar to A, but DS19(Tk-Ouar) cells that had beenirradiated with UV light at various intensities (abscissa) and incu-bated for 16 hr were fused with Me2SO-pulsed [1.8% (vol/vol), 16 hr](o) or untreated (o) BHK-21 cells. In other control experiments, fusionbetween UV-irradiated (25 J/m2) BHK-21 cells and similarly treatedDS19(Tk-Ouar) cells and between Me2SO-pulsed [1.8% (vol/vol), 16hr] BHK-21 cells and similarly treated DS19(Tk-Ouar) cells gave 1.5%and 0.7% benzidine-positive cells, respectively.

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FIG. 4. Effect of UV irradiationthroid differentiation in fusion-betwe(A) FM3A-cells that had been irradiat(abscissa) and then incubated for 16 h[1.8% (vol/vol), 16 hr] (.) or untreatecell fusion, the cells were incubated ibain (1.5 mM) for 5 days and benzidin(B) Similar to A, but DS19(Tk7Ouarwith UV at various intensities (abscisfused with Me2SO-pulsed [1.8% (vol/FM3A cells. In other control experimated (25 J/m2) FM3A cells and similaand between Me2SO-pulsed [1.8% (voltreated Friend cells gave 1.9% and Ospectively.

FIG. 5. Effect of UV irradiation and Me2SO treatment on ery-throid differentiation in fusions between two genetically markedFriend cells. (A) Double mutant Friend cells, DS19(TkhOuar), that hadbeen irradiated with UV at various intensities (abscissa) and then in-cubated for 16 hr were fused with Me2SO-pulsed [1.8% (vol/vol), 16 hr](o) or untreated (0) Friend (DS19) cells. After cell fusion, the cells wereincubated inHAT medium containing ouabain (1.5 mM) for 5 days andbenzidine-positive (Bk) cells were scored. (B) Similar to A, but DS19cells that had been irradiated with UV at various intensities (abscissa)and incubated for 16 hr were fused with Me2SO-pulsed [1.8% (vol/vol),16 hr] (.) or untreated (o) DS19(Tk-Ouar) cells. In other control ex-periments, fusion between UV-irradiated (25 J/m2) DS19(Tk~Ouar)and similarly treated DS19 cells andbetween Me2SO-pulsed [1.8% (vol/vol), 16 hr] DS19(Tk-Ouar) and similarly treated DS19 cells gave 4.4%and 0.9% benzidine-positive cells, respectively.

distribution of the reaction among various kinds of mammalian20 cells. Furthermore, in contrast to the Me2SO-induced reac-

tion(s), the UV-induced reaction(s) or the reaction product(s) hasB a trans-acting nature that facilitates or acts in concert with the

reaction(s) induced by Me2SO for erythroid differentiation. Onthe other hand, the Me2SO-induced reaction(s) is specific toFriend cells and is likely cis-acting and therefore cannot be sub-stituted for by the reaction(s) (or reaction products), if any, in-duced in other nonerythroid cells. In any event, our results have

10 - demonstrated that erythroid differentiation can be induced in-directly in Friend cells after cell fusion with nonerythroid cellsthat had been treated by UV light or other agents that affectDNA metabolism.One of the interesting but unsettled problems in erythroid

differentiation in Friend cells is the mechanism of action of tu-mor promoters. In general, tumor promoters such as TPA in-hibit erythroid differentiation induced by Me2SO or other in-

0 1 l20 30 ducing agents (19). By employing the cell fusion technique, we

VI J/m2 attempted to determine the site of the action of tumor pro-moters. Friend cells, DS19(Tk-) and DS19(Hprt-), were

and Me2SO treatment on ery- treated with UV or Me2SO and incubated in the presence orDen FM3A cells and Friend cells. absence of TPA before cell fusion. As seen in Table 2, whereas;edwith UVat various intensities Friend cells that had been irradiated by UV and incubated withir were fused with Me SO-pulsed *1 1 *.1ldr were fusedwithMr)e2 lsed TPA induced erythroid differentiation after cell fusion within HAT medium containing oua- Me2SO-exposed cells, the cells exposed to Me2SO in the pres-Le-positive(Bm ) cells were scored. ence ofTPA did not induce erythroid differentiation after fusion') cells that had been irradiated with UV-irradiated cells. These results clearly indicate that the3sa) and incubated for 16 hr were reaction step affected by TPA in erythroid differentiation inNvol), 16 hr] (.) or untreated (o) Friend cells is not involved in the reactions induced by UV butients, fusion between UV-irradi-irly treated DS19(TkOuar) cells is related to the reactions induced by Me2SO.[/vol), 16 hr] FM3A and similarly The results presented in this paper have shed some light on.9% benzidine-positive cells, re- the mechanism oferythroid differentiation in Friend cells. Most

importantly, our results strongly suggest that two fundamentally

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214 Cell Biology: Nomura and Oishi

Table 2. Effect of TPA treatment before cell fusion on erythroiddifferentiation

DS19(Tk-) DS19(Hprt-)TPA, TPA, Bendizine-positive

Treatment ng/ml Treatment ng/ml cells, %Me2SO 0 UV 0 31.5Me2SO 10 UV 0 4.1Me2SO 50 UV 0 3.7'Me2SO 0 UV 10 28.8Me2SO 0 UV 50 30.9

UV 0 Me2SO 0 28.8UV 10 Me2SO 0 23.6UV 50 Me2SO 0 29.1UV 0 Me2SO 10 4.4UV 0 Me2SO 50 2.9

During incubation (16 hr) of Friend cells (Tk- or Hprt-) after UVirradiation (25 J/m2) or incubation (16 hr) with 1.8% (vol/vol) Me2SObefore cell fusion, TPA (0, 10, or 50 ng/ml) was included in the medium.The cells were then fused. Benzidine-positive cells were scored on the5th day.

distinct intracellular reactions are involved in erythroid differ-entiation in Friend cells in vitro. As described above, one re-action is inducible by DNA-damaging agents, is nonspecific toFriend cells, and is trans-acting. The other reaction is specificto Friend cells, is likely to be cis-acting, and involves a sitewhere tumor promoters act. The effect of tumor promoters onthe latter reaction suggests that the reaction is membrane me-diated, at least in its early stage. On the other hand, these re-sults also have raised several questions concerning the effectofDNA damage on erythroid differentiation. For example, howdoes DNA damage affect or potentiate erythroid differentia-tion? Damage of chromosomal DNA by DNA-damaging agentssuch as UV may disturb or even inhibit cellular DNA replica-tion. This may trigger "SOS-type" reactions that affect cell di-vision or recombination capacity as observed in bacteria (20).Under such conditions, Friend cells acquire a potential to dif-ferentiate and do differentiate when appropriate transmem-brane signals are given.The results presented here also suggest that most of the

known inducing agents-such as Me2SO, butyric acid, hexa-methylenebisacetamide, and others-have dual effects onFriend cells. One is to create some specific signal through thecell membrane and the other is to damage DNA or affect DNA

metabolism. Experiments by Scher and Friend (11) and Teradaet al (12) in fact demonstrated that Me2SO causes damage (scis-sions) in DNA upon prolonged incubation of the Friend cellswith the drug. It is likely that agents that are capable ofcausingthese two kinds of specific effects are now classified as the in-ducing agents for erythroid differentiation in Friend cells.'The authors thank Ms. M. Harada for her excellent editing of the

manuscript and Dr. G. M. Pfaffenbach for critical reading of the manu-script. The authors are grateful to Dr. M. Terada for his advice andcomments and to Drs. D. Ayusawa, R. A. Riflind, G. Tamura, and M.Terada for providing us cell lines used in this study. This research wassupported by grants from the Nissan Science Foundation, the MinistryofEducation ofJapan, the Ministry ofHealth and Welfare ofJapan, andthe National Institutes of Health of the United States.

1. Friend, C., Patuleia, M. C. & de Harven, E. (1966) Natl. CancerInst. Monogr. 22, 505-520.

2. Friend, C., Scher, W., Holland, J. & Sato, T. (1971) Proc. NatlAcad. Sci. USA 68, 378-382.

3. Ross, J., Ikawa, Y. & Leder, P. (1972) Proc. Natl Acad. Sci. USA69, 3620-3623.

4. Orkin, S. H., Swan, D. & Leder, P. (1975) J. Bio. Chem. 250,8753-8760.

5. Sassa, S., Granick, S., Chang, C. & Kappas, A. (1975) in Eryth-ropoiesis, eds. Nakano, K., Fisher, J. W. & Tanaka, F. (Univ. ofTokyo Press, Tokyo), pp. 383-396.

6. Kabat, D., Sherton, C. C., Evans, L. H., Bigley, R. & Koler, R.D. (1975) Cell 5, 331-338.

7. Leder, A. & Leder, P. (1975) Cell 5, 319-322.8. Takahashi, E., Yamada, M., Saito, M., Kuboyama, M. & Ogasa,

K. (1975) Gann 66, 577-580.9. Reuben, R. C., Wife, R. L., Breslow, R., Rifkind, R. A. &

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Bibl Haematol (Basel) 39, 943-959.11. Scher, W. & Friend, C. (1978) Cancer Res. 38, 841-849.12. Terada, M., Nudel, U., Fibach, E., Rifkind, R. A. & Marks, P.

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