MOLECULAR AND CELLULAR BIOLOGY, Aug. 1988, p. 3311-3315 Vol. 8, No. 80270-7306/88/083311-05$02.00/0Copyright © 1988, American Society for Microbiology

Effect of Mitochondrial Protein Synthesis Inhibitors on ErythroidDifferentiation of Mouse Erythroleukemia (Friend) Cells

TOMOKO KANEKO, TOSHIO WATANABE, AND MICHIO OISHI*Institute ofApplied Microbiology, University of Tokyo, Bunkyo 113, Tokyo, Japan

Received 1 February 1988/Accepted 19 May 1988

When mouse erythroleukemia (MEL) cells were incubated in the presence of chloramphenicol (a specfficinhibitor for mitochondrial protein synthesis) during the early stage of in vitro erythroid differentiation, thenumber of induced erythroid cells was greatly reduced. By use of cell fusion between two genetically markedMEL cells, this finding was further investigated. We found that the drug, along with other agents which inhibitmitochondrial protein synthesis, blocked the induction and turnover of the DMSO-inducible intracellular-erythroid-inducing activity (differentiation-inducing factor II) in a manner similar to that of cycloheximide, aninhibitor for nuclear protein synthesis. The inhibitory effect was confirmed by directly assaying differentiation-inducing factor II in the cell extracts. These results strongly suggest that mitochondrial protein synthesis isclosely associated with in vitro erythroid differentiation of MEL cells.

Mouse erythroleukemia (MEL) cells (2) undergo dramaticirreversible changes in their morphological and biochemicalcharacteristics when the cells are exposed to a variety ofinducing agents (3, 6, 12, 13). Since the changes are quitesimilar to in vivo erythroid differentiation in many aspects,such as accumulation of hemoglobin in the cells, the induc-tion process has been studied extensively as a model forterminal differentiation of hematopoietic cells. In previouscell fusion experiments, we demonstrated that the in vitroerythroid differentiation of mouse erythroleukemia (MEL)cells is a synergistic result of two independent intracellularreactions (5, 8). The first reaction is associated with inhibi-tion ofDNA replication (or cell division, as a consequence),and the second one is derived from a transmembrane signalgenerated by erythroid-inducing agents, such as dimethylsulfoxide (DMSO) (3) or hexamethylenebisacetamide(HMBA) (12). The induction of the latter reaction apparentlyaccompanies de novo nuclear protein synthesis, since cyclo-heximide completely blocks the induction (5). These andsubsequent cytoplast fusion experiments (14) strongly sug-gested that DMSO (or HMBA) induces a trans-acting factorwhich triggers erythroid differentiation in coordination withthe other factor induced by inhibition of DNA replication.More recently, we have demonstrated the presence of twoactivities in cell extracts which correspond to these factors;differentiation-inducing factor I (DIF-I), responsible for thefirst reaction (9), and differentiation-inducing factor II (DIF-II), responsible for the second DMSO- or HMBA-induciblereaction (15).While testing a number of agents for their effects on

erythroid differentiation, we found that inhibitors of mito-chondrial protein synthesis inhibited the induction of differ-entiation. To confirm the effect, we investigated the inhibi-tion process by cell fusion between two genetically markedMEL cells. The results show that inhibitors for mitochon-drial protein synthesis, such as chloramphenicol or tetracy-cline, blocked erythroid differentiation by inhibiting theprocess leading to the induction of DIF-IT. The inhibitoryeffect did not exhibit a lag period, and the inhibitors alsoinhibited the turnover of DIF-II. The effect was confirmedby directly examining the effect of chloramphenicol on the

* Corresponding author.

appearance of DIF-II in the cell extracts from DMSO-treatedMEL cells. The significance of the finding is discussed.

MATERIALS AND METHODSMaterials. Thymidine, aminopterine, hypoxanthine, cy-

cloheximide, chloramphenicol, and tetracycline were pur-chased from Sigma Chemical Co. (St. Louis, Mo.). Polyeth-ylene glycol 6000 was obtained from J. T. Baker ChemicalCo. (Phillipsburg, N.J.). Minimal essential medium (MEM)was purchased from Nissui Seiyaku (Tokyo, Japan). Ham'snutrient mixture F12 and Dulbecco modified Eagle mediumwere obtained from Sigma. Fetal calf serum (FCS) wasobtained from Flow Laboratories, Inc. (McLean, Va.) andUnited Biotechnologies (Tokyo, Japan). All the other chem-icals used in this study were reagent grade.

Cells. A MEL cell line (DS19) was obtained from M.Terada. This cell line was derived from cell line 745. Amutant of DS19 (Tk- Hprt+) which lacks functional thymi-dine kinase was a generous gift from R. A. Rifkind. Anothermutant of DS19, DS19 (Tk+ Hprt-), presumably having adefective hypoxanthine phosphoribosyltransferase, was iso-lated in this laboratory from N-methyl-N'-nitro-N-nitroso-guanidine-treated DS19 cells as a clone resistant to 6-thioguanine at 15 ,ug/ml. A MEL cell line, 11A2, whichgrows in F12-Dulbecco modified Eagle medium containing alow concentration (1%) of FCS and which was used as asource of DIF-II, was established in this laboratory aftersuccessive adaptations of DS19 in the low-serum medium.

Cell culture. For cell fusion experiments, MEL cells werecultured in MEM supplemented with 12% heat-inactivatedFCS. The medium (HAT medium) used for the selection ofthe fused cells contained hypoxanthine (0.1 mM), aminop-terin (0.4 ,uM), thymidine (16 ,uM), and glycine (3 ,M) inMEM supplemented with 12% FCS. All the cultures wereincubated in plastic petri dishes (60 by 15 mm, Falcon;Becton Dickinson Labware, Oxnard, Calif.) or multiwellplates (24 wells, Falcon) at 37°C in a humidified atmospherecontaining 5% CO2 in air. For the preparation of DIF-II,MEL 11A2 cells were cultured in F12-Dulbecco modifiedEagle medium DMEM (1:1) supplemented with 1% FCS.The cells were grown in 10-liter spinner flasks at 37°C.UV irradiation. Confluently grown cells (1 x 106 cells per

ml) were collected by centrifugation (800 x g, 5 min) at room

3311

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 07

Jan

uary

202

2 by

122

.103

.105

.166

.

3312 KANEKO ET AL.

temperature and suspended in phosphate-buffered saline(PBS) (137 mM NaCl, 4.2 mM KCl, 9.6 mM Na,HPO4, 1.1mM KH2PO4) at a final cell density of 5 x 106 cells per ml.The cell suspension (5 ml) was transferred to a plastic petridish (60 by 12 mm) and irradiated under a Toshiba GL15(15-W) germicidal UV lamp to give a UV intensity of 25 J/m2.After irradiation, the cells were collected by centrifugationand suspended (7 x 105 cells per ml) in MEM supplementedwith FCS (12%), which consisted of a mixture of conditioned(used for cell growth) and fresh medium at a ratio of 2:1, forfurther incubation (24 h) at 37°C.

Cell fusion and selection. Cell fusion was performed by aprocedure of Pontecorvo (11) with modifications. The cells(106 of each type of cell) were first mixed and centrifuged(800 x g, 5 min) at room temperature. After the supernatantwas removed, the pellet was mixed with 0.2 ml of polyeth-ylene glycol 6000 (50%, wt/wt) and kept at room temperaturefor 2 min. MEM (1 ml) was then added, and after gentlemixing, the cell mixture was left at room temperature foranother 3 min. The cells were then diluted with 4 ml of MEMsupplemented with CS (10%), centrifuged (500 x g, 5 min),and suspended in 1 ml of MEM supplemented with FCS(12%). To this cell suspension, 25 Ill of 20x HAT solutionwas added, and the cell suspension was incubated for 5 daysat 37°C. The efficiency of cell fusion between the twogenetically marked cells was approximately 1% of the cellspresent at the time of fusion.

Assay of erythroid differentiation. Hemoglobin accumula-tion, a characteristic of erythroid differentiation, was as-sayed by the method of Orkin et al. (10) by benzidinestaining of hemoglobin accumulated in the cells. In short, acell suspension (200 pl) was mixed with 20 pu1 of freshlyprepared benzidine solution (10:1 mixture of 0.2% 3,3-dimethoxybenzidine in 0.50 M acetic acid and 30% hydrogenperoxide), and stained cells were scored under a micro-scope. At least 600 cells were examined (in duplicate) at eachassay.

Assay of [35S]methionine incorporation into proteins. MEL(DS19, Tk- Hprt+) cells were grown in MEM (supplementedwith 12% FCS) with the methionine concentration adjustedto 100 p.M. At a cell density of 106/ml, the cells were treatedwith DMSO (1.8%, vol/vol) for 2 h, washed with PBS, andsuspended in the same volume (total, 10 ml) of MEM (12%FCS) without methionine. [35S]methionine (40 pLCi/ml) wasthen added, and incubation was continued at 37°C in a CO,incubator. At various times, samples (50 [l) were with-drawn, and trichloroacetic acid (20%, wt/vol)-insoluble frac-tions were counted in a scintillation counter after the mate-rial was collected on a glass filter (GF/C; Whatman, Inc.,Clifton, N.J.). Chloramphenicol or cycloheximide wasadded at the time of [35S]methionine addition.

Assay of DIF-II activity. DIF-II activity was assayed asdescribed previously (15) by using permeabilized MEL cells(7). In essence, MEL cells (11A2) were cultured in thepresence of DMSO (1.8%, vol/vol) for 6 h in 10-liter spinnerflasks, and after disruption of the cells with a homogenizer,the cytosol fraction (-100 mg of protein or more) wasapplied to a DEAE column. The eluate (50 mM NaCl) wasintroduced into the recipient permeabilized MEL cells whichhad been irradiated by UV light 24 h before. The benzidine-positive (B+) cells were counted 5 days later.

RESULTS

While screening a number of agents which specificallyaffect in vitro erythroid differentiation of MEL cells, we

0 50 100Chloramphenicol (jig/ml)

FIG. 1. Effect of chloramphenicol on the erythroid induction ofMEL cells. MEL cells (DS19) cultured to confluent growth werediluted with fresh medium to a cell density of 2 x 105 cells per ml andincubated in a plastic plate (24 weeks, 1.0 ml each) in the presenceof DMSO (1.8X. vol/vol) and the indicated concentrations ofchloramphenicol. After 48 h of incubation at 37°C, the cells werewashed twice with PBS, and incubation was continued in the freshmedium (without drugs) for another 72 h, when benzidine-positive(B+) cells were counted (for details, see Materials and Methods).

found that the presence of chloramphenicol during the earlystage of the differentiation process significantly inhibited theerythroid induction. Incubation of the cells with the drug forthe first 48 h after the addition of an inducer (DMSO), themost critical period for the cellular commitment to differen-tiation (4), reduced the number of benzidine-positive (B+)cells (an indication of hemoglobin accummulation in thecells) by over 90% (Fig. 1). At the same time, the drug alsoblocked the loss of viability, which is one of the character-istics of differentiated cells. In a typical experiment, whenerythroid differentiation was inhibited 93% by chloramphen-icol (87% B + cells in a sample of control cells versus 6% Bcells in the presence of chloramphenicol), the normalizedcolony-forming ability increased from 8% (control cells) to89% in the presence of the drug, indicating that cellularcommitment, not just hemoglobin accumulation, was in factinhibited. Such effects were not observed when chloram-phenicol was added at the later stage (48 to 120 h) of thedifferentiation (data not shown). The details of the experi-ments will be reported elsewhere (T. Watanabe, H. Oku-mura, S. Nomura, and M. Oishi, submitted for publication).To investigate the effect further, a series of cell fusion

experiments was performed using two types of geneticallymarked MEL cells. Previously, we reported that a relativelyshort-lived activity responsible for erythroid differentiationis induced following treatment of the cells with DMSO (orHMBA) (5). The activity was detected only when theDMSO-treated cells were fused with the cells which hadbeen treated with agents affecting DNA replication. Appar-ently, the DMSO-induced activity triggered erythroid differ-entiation in coordination with a factor generated by inhibi-tion of DNA replication. The induction of the activity isillustrated in Fig. 2. MEL cells (Tk- Hprt+) which had beenpulsed with DMSO (1.8%, vol/vol) for 2 h were incubated fordifferent times and fused with UV-irradiated MEL cells (Tk tHprt-). The cells were then incubated in a selection (HAT)medium, and the number of benzidine-positive cells amongthe surviving cells was scored after 5 days. The percentageof differentiated cells kept increasing up to 10 h of incubationafter the DMSO treatment. Further incubation resulted inthe decline of the differentiated cells. The induction of theactivity was not detected when the cells were fused withnonirradiated cells (Fig. 2), indicating that a synergisticreaction with the UV-induced activity was required for theDMSO-induced activity to materialize as the erythroid-

MOL. CELL. BIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 07

Jan

uary

202

2 by

122

.103

.105

.166

.

INHIBITION OF ERYTHROID DIFFERENTIATION 3313

ae

m

aR

co~

m

C D

10 20 30 40

Time, hrFIG. 2. Induction of the activity by DMSO revealed by cell

fusion with UV-irradiated cells. MEL cells (DS19, Tk- Hprt+) weregrown to a cell density of 2 x 106 cells per ml. The cells were thendiluted (1:1), and DMSO was added to a final concentration of 1.8%,vol/vol. After 2 h of incubation, the cells were centrifuged (800 x g,5 min), washed twice with PBS, suspended in mnedium (106 cells perml), and further incubated at 37°C. At different times, 1.0-mlsamples were withdrawn and mixed with 106 cells which had beenUV irradiated at 24 h before cell fusion (see below). For UV-irradiated cells, MEL cells (DS19, Tk+ Hprt-) were irradiated withUV light (25 J/m2) and incubated further for 24 h at 37°C. As acontrol, cells were treated in the same way except that UV irradi-ation was omitted. These cells were then used for cell fusion withDMSO-treated cells. DMSO-treated cells fused with UV-irradiatedcells (0). DMSO-treated cells fused with control cells (0). After cellfusion, the cells were incubated in HAT medium for 5 days, andbenzidine-positive (B+) cells were counted (for details, see Materi-als and Methods).

inducing activity. Likewise, no increase of B+ cells wasdetected when the DMSO-pulsed cells were incubated with-out cell fusion (data not shown). As will be discussed, themolecular basis for the DMSO-induced activity is very likelyto be DIF-1I, which was recently demonstrated in cellextracts from DMSO-treated MEL cells (15).The induction of the DMSO-induced activity detected by

cell fusion was inhibited by cycloheximide (or emetin), aninhibitor for nuclear protein synthesis, suggesting that denovo nuclear protein synthesis is involved in the induction(5). When we used chloramphenicol instead of cyclohexi-mide, essentially the same inhibitory effect as that of cyclo-heximide was observed in respect to the induction of theactivity (Fig. 3). MEL cells were pulsed with DMSO (1.8%,vol/vol) for 2 h. After removal of DMSO, the cells wereincubated in the absence or presence of chloramphenicol (50p,g/ml). Vamples with chloramphenicol were withdrawn at10, 20, 30, and 40 h (Fig. 3) and immediately washed toremove the drug. The cells were further incubated, and aportion of each sample was subsequently fused with UV-irradiated cells at different time intervals, as was done for theexperiments shown in Fig. 1. The number of benzidine-positive cells among fused cells was scored 5 days later. Thepresence of chloramphenicol suppressed the induction of theactivity apparently without a significant lag period (Fig. 3).Removal of the drug immediately restored the induction ofthe activity for up to 20 h of incubation in the presence ofchloramphenicol. A similar phenomenon was observed withcycloheximide (5). The presence of chloramphenicol duringthe DMSO pulse had no effect (data not shown).The inhibitory effect of chloramphenicol on the induction

of the activity as a function of the drug concentration isshown in Fig. 4A. The maximum inhibition was approxi-

Time, hrFIG. 3. Effect of chloramphenicol on the induction of the activity

as a function of time of incubation with the drug. MEL cells (DS19,Tk- Hprt+) were grown to a cell density of 2 x 106 cells per ml. Thecells were then diluted (1:1), and DMSO was added to a finalconcentration of 1.8%, vol/vol. After 2 h of incubation, the DMSO-treated cells were centrifuged (800 x g, 5 min), washed twice withPBS, and suspended in FCS-MEM (106 cells per ml). The cells werethen incubated in the absence (0) or presence (@) of chloramphen-icol (50 ,ug/ml). Samples with chloramphenicol were withdrawn at10, 20, 30, and 40 h of incubation as indicated by arrows A, B, C,and D, respectively, washed twice with 5 ml of PBS each time toremove the drug, and incubated again in FCS-MEM. At intervals of2 to 3 h, the cells were fused with UV-irradiated cells (DS19, Tk+Hprt-) and incubated in HAT medium for 5 days. Benzidine-positive (B+) cells were then counted (for details, see Materials andMethods).

mately 75 to 80%, which was slightly less than that bycycloheximide, in which 85 to 90% inhibition was usuallyobserved. Besides chloramphenicol, other agents whichinhibit mitochondrial protein synthesis, including tetracy-cline (Fig. 4B) and erythromycin (data not shown), had aneffect similar to that of chloramphenicol.

Previously, we showed that the induced activity remainsactive for at least 10 h without a sign of decline whencycloheximide is added after full induction of the activity,

A B15

20-

10

010m ~~~~~~~5-

0 25 50 75 0 1 10 100Chloramphenicol, jig/ml Tetracycline, jig/ml

FIG. 4. Effect of chloramphenicol and tetracycline on the induc-tion of the differentiation-inducing activity. MEL cells (DS19, Tk-Hprt+) were grown to a cell density of 2 x 106 cells per ml. The cellswere then diluted (1:1), and DMSO was added to a final concentra-tion of 1.8%, vol/vol. After 2 h of incubation, the cells werecentrifuged (800 x g, 5 min), washed twice with PBS, suspended inFCS-MEM, and incubated for 6 h with chloramphenicol (A) ortetracycline (B) at various concentrations. The cells were then fusedwith the cells which had been irradiated with UV 24 h before. Afterfusion, the cells were incubated in HAT medium for 5 days, andbenzidine-positive (B+) cells were counted (for details, see Materi-als and Methods).

VOL. 8, 1988

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 07

Jan

uary

202

2 by

122

.103

.105

.166

.

3314 KANEKO ET AL.

20

0

0+

10

wo;$- . N ..

0._0_0

:00

10 10 20 30DMSO

Time,hrFIG. 5. Effect of chloramphenicol on the turnover of the activity

induced by DMSO. MEL cells (DS19, Tk- Hprt+) were grown to acell density of 2 x 106 cells per ml. The cells were then diluted (1:1),and DMSO was added to a final concentration of 1.8%, vol/vol.After 2 h of incubation, the cells were centrifuged (800 x g, 5 min),washed twice with PBS, and suspended in FCS-MEM (106 cells perml). At various times, 1-ml samples were withdrawn and fused withthe cells which had been UV irradiated 24 h before. At 6 h after theremoval of DMSO (arrow), chloramphenicol (50 ,ug/ml) was addedto the sample (0, no chloramphenicol added). Chloramphenicol wasthen removed at 6 (A) and 12 (-) h. At various times, a portion ofeach sample was fused with UV-irradiated cells. The fused cellswere incubated in HAT medium for 5 days, and benzidine-positivecells (B+) were scored (for details, see Materials and Methods).

indicating that the induction as well as the turnover of theactivity is inhibited by cycloheximide (5). When chloram-phenicol was added at the peak of the induced activity, theactivity remained constant for at least 10 h and declined onlyafter the removal of chloramphenicol (Fig. 5). Without thedrug, the induced activity remained only transiently. Thus,the effects of chloramphenicol (tetracycline) on the inductionand turnover of the activity were essentially the same asthose of cycloheximide, suggesting that inhibition of proteinsynthesis, either in nuclei or mitochondria, exerts the sameeffects on the induction and turnover of the activity inducedby DMSO.The observed effects of chloramphenicol and other inhib-

itors for mitrochondrial protein synthesis did not seem tostem from general adverse effects of impaired mitochondrialfunctions due to the inhibition of mitochondrial proteinsynthesis because such effects are expected to occur onlyafter relatively long hours of exposure of the cells to theinhibitors. To confirm this, we examined the effect of chlor-amphenicol on the incorporation of [35S]methionine intoproteins under conditions similar to those of the cell fusionexperiments. No detectable inhibitory effect was observedfor at least the first 8 h of incubation, a period much longerthan that required to show the effect of the drug on theinduction and turnover of the activity (Fig. 6). These resultsimply that the effects of chloramphenicol are rather specificto the induction and turnover of the DMSO-induced activityand that mitochondrial protein synthesis is somehow in-volved in the induction process.

Recently, we reported a DMSO-inducible proteinaceousdifferentiation-inducing factor (DIF-II) to be present in thecell extracts of MEL cells (15). The factor triggers erythroiddifferentiation on introduction into undifferentiated MELcells, provided that the recipient cells are fully induced foranother differentiation-inducing factor (DIF-I) (9). Fromseveral criteria, we have concluded that the factor (DIF-IT) is

A

2 4 6 8

Time,hrFIG. 6. Effect of chloramphenicol and cycloheximide on the

incorporation of [35S]methionine into the acid-insoluble fraction.MEL (DS19, Tk- Hprt+) cells were cultured and treated withDMSO. DMSO was removed, and the incorporation of [35S]methi-onine into the acid-insoluble fraction was assayed as described inMaterials and Methods. Symbols: 0, control; *, chloramphenicol(50 ,ug/ml); and A, cycloheximide (0.5 p.g/ml).

identical or closely related to the DMSO-induced activityrevealed by the cell fusion experiments (15). To examinewhether the induction of DIF-IT activity in the extracts isalso affected by chloramphenicol, MEL cells were treatedwith DMSO and incubated in the presence of chloramphen-icol. The extracts were prepared 6 h later, and DIF-I1activity was assayed after a stepwise DEAE column chro-matography of the extracts. DIF-I1 activity, which is usuallyeluted at 50 mM NaCl from the DEAE column, was greatlyreduced in the sample prepared from the cells incubated inthe presence of chloramphenicol (Fig. 7). On the other hand,the induction of DIF-I was not inhibited by chloramphenicol(data not shown). These results are consistent with those ofthe cell fusion experiments described above and furtherindicate that inhibition of mitochondrial protein synthesisspecifically inhibits the induction of the DMSO-induceddifferentiation-inducing activity.

DISCUSSION

Use of cell fusion designed for analysis of the mechanismof in vitro erythroid differentiation provided opportunities tocharacterize the early induction process (5, 8). The sametechnique has made it possible to investigate effects ofvarious agents on the differentiation process more directlythan by conventional procedures, without possible compli-cations which might result from secondary effects caused bythe agents. Taking advantage of this new procedure, wefound that chloramphenicol or other agents which inhibitmitochondrial protein synthesis blocked erythroid differen-tiation in MEL cells. The effect is apparently due to inhibi-tion of the induction of the DMSO-induced activity (DIF-II)which is responsible for MEL cell differentiation.There are several reports implicating involvement of cy-

toplasmic elements, mostly mitochondrial genes, in specificgene expression. Fischer-Lindahl and her associates (1)demonstrated that a mitochondrial gene is directly involvedin the expression of a mouse surface antigen. In this case,the expression of the antigen is controlled by the two nucleargenes and one mitochondrial gene. We have several, al-though quite speculative, explanations of the effect of chlor-

MOL. CELL. BIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 07

Jan

uary

202

2 by

122

.103

.105

.166

.

INHIBITION OF ERYTHROID DIFFERENTIATION 3315

B50 mM 150 mM 250 mM

$ t t10 12

EC

5 20X

10 20"Fraction No.

FIG. 7. Effect of chloramphenicol on the induction of DIF-II.MEL cells (11A2) (approximately 1010 cells; total, 10 liters) wereincubated for 6 h in the presence of DMSO (1.8%, vol/vol) (A) orDMSO (1.8%, vol/vol) plus chloramphenicol (100 1Lg/ml) (B). Thecytosol fraction (80 mg of protein) was applied to a DEAE column(1.2 by 4.0 cm) and eluted in a stepwise manner with 15 ml each of50 mM, 150 mM, and 250 mM NaCl in basal buffer (20 mM Trishydrochloride, pH 7.5, 10% glycerol, vol/vol, 0.25 mM dithiothrei-tol). Samples (20 ,ul) of each fraction (2.6 ml) were assayed forDIF-II activity by using as recipients DS19 cells that had beenirradiated with UV light 24 h before and made permeable asdescribed previously (7, 15). DIF-II activity (0) is shown as thepercentage of benzidine-positive (B+) cells induced. Protein concen-trations (A) in each fraction are also shown.

amphenicol on erythroid differentiation. The most straight-forward one is that the DMSO-induced activity, DIF-II, isderived from a gene in mitochondria. Although the molecu-lar nature of DIF-II is still subject to investigation, recentresults have shown that the molecular size of DIF-II is quitelarge, over 250,000 daltons, and that it probably consists ofsubunit protein molecules (15). Therefore, it is possible forone of the subunits of DIF-II to be coded by a mitochondrialgene and the rest of them to be coded by nuclear genes.Essentially, the fact that DIF-II induction is similarly af-fected by inhibitors of nuclear protein synthesis (5) and bychloramphenicol (tetracycline) supports this possibility. Onthe other hand, a more indirect, yet unidentified, role ofmitochondrial protein synthesis in regulating cellular differ-entiation may be considered. For example, inhibition ofprotein synthesis, either in nuclei or mitochondria, maygenerate a signal which negatively controls gene expressionspecific to the commitment of MEL cell differentiation.Finally, it is also possible that the early cellular processleading to the induction of DIF-II is extremely sensitive to asubtle change in the mitochondrial functions, for example, aslight drop in ATP concentration, although it may not affectoverall cellular functions.

ACKNOWLEDGMENTS

We thank Y. Okamoto for her assistance in editing the manuscriptand S. Nomura and H. Okumura for their helpful discussions.

This work was supported by a grant from the Japanese Ministry ofEducation.

LITERATURE CITED1. Fischer-Lindahl, K., B. Hausman, P. J. Robinson, J.-L. Gounet,

D. C. Wharton, and H. Winking. 1986. Mta, the maternallytransmitted antigen, is determined jointly by the chromosomalHmt and the extrachromosomal Mtf genes. J. Exp. Med. 163:334-346.

2. Friend, C., M. C. Patuleia, and E. DeHarven. 1966. Erythrocyticmaturation in vitro of murine (Friend) virus-induced leukemiacells. Natl. Cancer Inst. Monogr. 228:505-520.

3. Friend, C., W. Scher, J. G. Holland, and T. Sato. 1971.Hemoglobin synthesis in murine virus-induced leukemia cells invitro: stimulation of erythroid differentiation by dimethyl sulf-oxide. Proc. Natl. Acad. Sci. USA 68:378-382.

4. Guseila, J., R. Geler, R. Clarke, V. Weeks, and D. Housman.1976. Commitment to erythroid differentiation by Friend eryth-roleukemia cells: a stochastic study. Cell 9:221-229.

5. Kaneko, T., S. Nomura, and M. Oishi. 1984. Early eventsleading to erythroid differentiation in mouse Friend cells re-vealed by cell fusion experiments. Cancer Res. 44:1756-1760.

6. Leder, A., and P. Leder. 1975. Butyric acid, a potent inducer oferythroid differentiation in cultured erythroleukemia cells. Cell5:319-322.

7. Nomura, S., T. Kamiya, and M. Oishi. 1986. A procedure tointroduce protein molecules into living mammalian cells. Exp.Cell Res. 163:434 444.

8. Nomura, S., and M. Oishi. 1983. Indirect induction of erythroiddifferentiation in mouse Friend cells: evidence for two intracel-lular reactions involved in the differentiation. Proc. Natl. Acad.Sci. USA 80:210-214.

9. Nomura, S., S. Yamagoe, T. Kamiya, and M. Oishi. 1986. Anintracellular factor that induces erythroid differentiation inmouse erythroleukemia (Friend) cells. Cell 44:663-669.

10. Orkin, S. H., F. I. Harosi, and P. Leder. 1975. Differentiation inerythroleukemia cells and their somatic hybrids. Proc. Natl.Acad. Sci. USA 72:98-102.

11. Pontecorvo, G. 1975. Production of mammalian somatic cellhybrids by means of polyethylene glycol treatment. SomaticCell Genet. 1:397-400.

12. Reuben, R. C., R. L. Wife, R. Bresiow, R. A. Rifkind, and P. A.Marks. 1976. A new group of inducers of differentiation inmurine erythroleukemia cells. Proc. Natl. Acad. Sci. USA 73:862-866.

13. Takahashi, E., M. Yamada, M. Saito, M. Kuboyama, and K.Ogasa. 1975. Differentiation of cultured Friend leukemia cellsinduced by short-chain fatty acids. Gann 66:577-580.

14. Watanabe, T., S. Nomura, and M. Oishi. 1985. Induction oferythroid differentiation by cytoplast fusion in mouse erythro-leukemia (Friend) cells. Exp. Cell Res. 159:224-234.

15. Watanabe, T., and M. Oishi. 1987. Dimethyl sulfoxide-induciblecytoplasmic factor involved in erythroid differentiation inmouse erythroleukemia (Friend) cells. Proc. Natl. Acad. Sci.USA 84:6481-6485.

* 10

0

A50 mM 150 mM 250 mM

$ t $

10 20

VOL. 8, 1988

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 07

Jan

uary

202

2 by

122

.103

.105

.166

.