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Vol. 5, 743-751, July 1994 Cell Growth & Differentiation 743
Differentiation of WEHI-3B D� Myelomonocytic LeukemiaCells Induced by Ectopic Expression of theProtooncogene c-jun1
Jianming Li, Ivan King, and Alan C. Sartorelli2
Department of Pharmacology and Developmental Therapeutics Program,Comprehensive Cancer Center, Yale University School of Medicine,
New Haven, Connecticut 06510
Abstract
The product of the protooncogene c-jun is one of thecomponents of the AP-1 transcription factor complex,which is involved in the control of cell proliferation anddifferentiation. To study the role of c-jun in leukemiacell growth and maturation, a plasmid (pMTJ1 1) wasconstructed that contained the rat c-jun complementaryDNA under the control of the human metallothioneinpromoter and the neo gene. Murine myelomonocyticWEHI-3B D� cells were transfected by electroporationwith the linearized pMTJ1 1 plasmid and subsequentlycloned in the presence of G-41 8. Exposure of theseclones to cadmium resulted in a high level of expressionof c-jun mRNA and protein, as demonstrated byNorthern hybridization and Western blotting. Whenthese clones were examined immediately after theirestablishment, expression of c-jun was accompanied bythe appearance of a mature phenotype in many clones,as measured by the reduction of nitroblue tetrazoliumand by the expression of Mac-i (CD1 1 b), a cell surfacemarker on differentiated cells. Morphological changesindicative of the differentiated state were also observedby staining. These findings indicate that expression ofc-jun is capable of initiating the differentiation of WEHI-3B D� cells in the absence of an external inducer ofmaturation. Furthermore, the expression of c-jun led toan enhancement of the induction of the differentiationof WEHI-3B D� cells by retinoic acid, suggesting aninvolvement of c-jun in the retinoic acid signaltransduction pathway.
Introduction
Hematopoiesis is characterized by the continuous turnoverof more than several hundreds of billions of blood cells perh. Functionally mature cells are generated through concom-itant proliferation and differentiation of plunipotent stemcells. The terminal differentiation of stem cells results in thecessation of growth and the formation of a variety of maturecell types, including macnophages and granulocytes. Therate of proliferation and differentiation of cells is carefullyregulated to maintain a steady state of hematopoiesis and to
modulate the equilibrium under conditions of stress. Dis-nuption of the regulatory machinery of this vital process at
any stage of maturation has severe consequences to life,
producing diseases such as leukemia.
In the process of myeloid differentiation, a series of bio-
chemical and morphological changes takes place through
an intrinsic genetic program; however, little is known aboutthe molecular mechanisms which control this program.
Several protooncogenes, particularly c-jun, c-los, c-myc,
and c-myb, have been implicated in the regulation of thecellular growth and differentiation of hematopoietic cells(1 -7). c-jun codes for a component ofthe AP-1 transcriptionfactor complex (8-i 0). The products of the c-jun gene andtwo related genes, jun-B and jun-D (1 1 -1 3), can form ho-
modimens on hetenodimens with the products of the los
family, including c-los, los-B, and fra-1, through leucine-
zipper binding (1 4-i 7). These complexes bind to a hep-tamenic DNA consensus sequence, TGAG/CTCA (AP-i
binding site), and through this binding can both activateand repress transcription of target genes containing theAP-i site. During monocytic differentiation of U937,
THP-1 , and HL-60 leukemia cells induced by TPA,3 oka-daic acid, bryostatmn, on 1,25-dihydnoxyvitamin D3, c-junexpression is rapidly up-regulated (1 , 7, 1 8). The increase in
c-jun expression in differentiating U937 cells is inhibited bydexamethasone, which also blocks the appearance of themonocytic phenotype, implying that c-jun expression isrequired for monocytic maturation (1 9). c-jun expression isalso activated when Mi leukemic myeloblasts and normalmyeloblasts are induced to differentiate to granulocytes onmacnophages by mouse lung conditioned medium, inter-leukin 6, granulocyte colony-stimulating factor, on macno-phage colony-stimulating factor (20) and when human pe-nipheral blood granulocytes are activated by TPA (6).Treatment of leukemia cells with several anticancerdrugs, such as anabinofuranosylcytosine, diamminedi-
chloroplatinum, on camptothecin, however, induces ex-pression of the c-jun gene but causes inhibition of growthand differentiation (21-24).
To further define the role of the expression of c-jun in
myeloid differentiation, we have constructed a c-jun ex-pression plasm id, transfected muni ne myelomonocyticWEHI-3B D’ leukemia cells, which can undergo both gran-ulocytic and monocytic differentiation, with this plasmidand measured the effects of the expression of c-jun oncellular differentiation. The results of these experimentsshowed that c-jun expression is capable of initiating thedifferentiation of WEHI-3B D� leukemia cells.
Received 10/4/93; revised 3/25/94; accepted 4/25/94.1 This report was supported in part by USPHS Grant CA-02817 from theNational Cancer Institute and by Grant 92 from the American Institute for
Cancer Research.
2 To whom requests for reprints should be addressed, at Department ofPharmacology, Yale University School of Medicine, 333 Cedar Street, New
Haven, CT 06510.
3 The abbreviations used are: TPA,1 2-O-tetradecanoylphorbol 1 3-acetate;FBS, fetal bovine serum; NBT, nitroblue tetrazolium; FITC, fluorescein iso-
thiocyanate; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate.
WD�
WD�V
WD� F
WD�J
2
3
4
5
6
2
3
4
5
6
7
8
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
744 WEHI-3B D� Cell Differentiation by c-jun
Results
WEHI-3B D� cells were transfected with either the c-jun onthe c-los expression plasmid, pMTJ1 1 on p75/i 5, respec-tively. Both c-jun and c-los genes were placed into the samevector, which contains an inducible metallothionein pro-moten, that was used as a control in the transfection exper-iments to investigate the effects of the expression of thec-jun and c-los genes on cellular differentiation. Trans-fected cells were selected with G-4i8, and single cellclones were obtained that were cultivated in modifiedMcCoy’s SA medium containing 1 5% FBS in the presenceon absence of 80 �M CdCl2 to induce the expression of c-junon c-los. The effects of the expression of c-jun and c-los
were determined after 3 days by measuring the ability ofcells to reduce NBT. In the presence of CdCl2, about 50%
of c-jun clones expressed a significant degree of diffenenti-ation (Table 1 ), with some of the clones having more than90% of cells exhibiting a mature phenotype. This level ofdifferentiation was not obtainable even with retinoic acid, apotent inducer of the granulocytic differentiation ofthis cellline (25). In contrast, parental WEHI-3B D� cells, six vector-tnansfected clones, and eight c-los transfected clones didnot exhibit any significant maturation.
The appearance of a mature phenotype in c-jun-trans-fected WEHI-3B D� cells was further confirmed by theexpression of Mac-i , a cell surface marker of differentia-tion. Cells were stained for immunofluorescence with FITC-conjugated anti-Mac-i antibody and analyzed by flow cy-
tometny (Fig. 1 ). No apparent difference was seen between
antibody-stained cells and unstained cells in the presenceon absence of cadmium with parental WEHI-3B D� cells.Induction of the differentiation of parental cells by 7 �M
netinoic acid (the optimum concentration of the retinoid toinduce the differentiation of this cell line) resulted in a highlevel of expression of Mac-i with an 8.3-fold increase inrelative fluorescence intensity. The c-jun-tnansfected cloneWD�J32, when stained with the antibody, also exhibited adifferentiated phenotype with a 1 7.3-fold increase in therelative fluorescence intensity in the presence of CdCI2. Therelative fluorescence intensity of these cells was 1 0.2-foldhigher than the control in the absence of CdCl2, a findingconsistent with the observation that NBT-positive cells werepresent in the absence of CdCI2 and that more NBT-positivecells were obtained when CdCI2 was included in the assay(Table 1 ). Thus, the results demonstrated that transfection ofWEHI-3B D� cells with c-jun but not c-los initiated thematuration process.
To determine whether the differentiation that occurredfollowing the transfection of c-jun resulted from the expres-sion of c-jun, we measured the steady state levels of c-junmRNA and protein in transfected cells. Cells were grown inmodified McCoy’s 5A medium containing 1 5% FBS in theabsence and presence of CdCI2 and collected for measure-ment of c-jun RNA and protein by Northern hybridizationand Western blotting, respectively. In the absence of CdCl2,relatively high basal levels of expression of c-jun wereobserved by Northern and Western blotting (data notshown), indicating that the metallothionein promoter wasleaky. Others have reported that the human metallothioneinpromoter is leaky (26, 27). The high basal level of c-junexpression presumably accounts for the high level of spon-taneous differentiation by c-jun-transfected WEHI-3B D�cells in the absence of CdCl2 (Table 1). To better understandthe effects of the expression of c-jun on the differentiation
Table 1 Differentiation of WEHI-3B D� cells transfected with c-los orc-jun protooncogenes
Cell lined Cloneb
___NBT-positive cells l”/o)
No .. Cadmium
cadmium
1 3
0 1
1 1
0 0
1 0
0 2
0 0
1 1
1 1
0 0
1 2
2 0
0 0
2 4
1 3
2 0
0 3
0 2
3 34
86 92
75 89
0 7
1 2
87 98
23 56
11 31
0 2
1 3
1 1
10 30
2 1
13 33
7 16
1 2
5 7
0 5
3 20
9 17
86 92
0 16
5 3
2 4
0 2
15 35
0 6
31 1 3
32 43 75
33 3 17
� parental WEHI-3B D� cells; WD�V, WD�F, and WD�J are
WEHI-3B D#{176}cells transfected with vector alone, p75/i 5 (c-los), and pMTJ1 1
(c-fun), respectively.b Clones were obtained by single-cell sorting using a FACS IV flow cytometerafter enrichment for transfected cells by G-41 8 selection and expansion ofeach single cell in multiwell plates.C Cells treated with 80 �M CdCl2 for 3 days.
process, we adapted c-jun-tnansfected cells to grow inX-VIVO-1 5 serum-free medium shortly after transfection inan effort to lower the basal expression of c-jun. Fig. 2Ashows the expression of c-jun mRNA driven by the metal-lothionein promoter in serum-free medium in the presenceand absence of 10 �M CdCl2. Among six c-jun-tnansfected
A C
WEHI-3B D�, no CdCI2
500�
a b
a, without Mac-i antibodyb, with Mac-I antibody
250’
��3�J32 no CdC12
500’
0
a, without Mac-i antibodyb. with Mac-l antibody
250�
.�
z
.�
z
b
100 10’ 10� l0�
Fluorescence intensity
B
750
.�
z
=
I
0 100 1& 10� 10�
Fluorescence Intensity
WEHI-3B D�. CdC12
a, without Mac-i antibodyb, with Mac-i antibody
750�
0
WD�’J32, CdCI2
a, without Mac-i antibody
b. with Mac-I antibody
b
100 101
Fluorescence Intensity
102
E
0 io#{176} lOl io� io�Fluorescence Intensity
.� 500�
z=
WEHI-3B D�. retinoic *id
a, without Mac-i antibodyb. with Mac-I antibody
250�
b
0 uP 102 10�
Cell Growth & Differentiation 745
101
fluorescence Intensity
Fig. 1. The effects of c-jun expression on the levels of Mac-i . Cells were grown in the presence or absence of 50 �M CdCl2 for 3 days and stained forimmunofluorescence with FITC-conjugated anti-c-Jun antibody as described in “Materials and Methods.” In all panels, a and b represent antibody unstained and
stained cells, respectively. WEHI-3B D� cells (A and B) and the c-jun-transfected WD�J32 cells (Cand D) were cultured in the absence (A and C) and presence
(B and D) of CdCl2. Retinoic acid (7 psi)-treated WEHI-3B D� cells (F) were used as a positive control.
A
Clone D’SFVl DSFJ1 D’SFJ2 D’SFJ3 D’SFJ4
CdC�2 - + - + - + - + -
1 2 3 4 5 6 7 8 9
28S
18S
B
Clone D�SFV1 D�FI D�F2 D’F3 D’F4 IYF5 D�F6
CdCt� - + - + - + - + - + - + - +
I 2 3 4 5 6 7 8 9 10 II 12 13 14
28S
: ,�
18S �
- �� .
��ic-1 �
�- c-los
746 WEHI-3B D ‘ Cell Differentiation by c-jun
D1S1�J5 D’SFJ6
+ - + - +
10 11 12 13 14
Fig. 2. Expression of c-jun and� �- C-jun c-losmRNAs ii) I)MTIt1 and p75/15-transfected WEHI-3B 0’ cells.Cells were grosvn br 2 days inX-VIVO-1 S seruni-Iree medium inthe absence or the presenoe of10 p.’,i CdCl,. Total cellular RNAs
svere isolated as described in“Materials and Methods” andhybridized with �P-labeleol c-junand c-los probes. A. the c-jun-
transtected clones, D ‘ SF11 , 2, 3, 4.
5. and 6; B. the c-tos-transfectedclones DF1, 2, 3. 4, 5, and 6.Vector-translected WEHI-3B 0’
cells )D ‘ SFV1 ) are used as thecontrol. Arrows, exogenously cx-pressed c-jun and c-tos mRNAs.
clones, five exhibited a significantly higher expression ofc-jun when cadmium was included in the medium; theother clone exhibited a level of c-jun expression in theabsence of CdCl2 equivalent to that occurring in the pres-ence of the inducer. These clones were also used to meas-ure the level of c-Jun protein by Western blotting (Fig. 3A).
In the presence of iO �M CdCI,, a significant increase inc-Jun protein was observed in five of the clones. It is notedthat clone D�SFJ2 did not show a significant increase inc-Jun protein, although an increase in the level of mRNAwas observed in this clone in the presence of CdCl2. Incontrast, clone D*SFJ5 exhibited a significant increase inc-Jun protein with no apparent increase in mRNA, indicat-ing that c-jun expression is not only regulated at the tran-scniptional level but is also controlled at the translationallevel. Cells transfected with vector alone had no detectablec-Jun protein, even in the presence of cadmium. Thisfinding implies that the expression of c-Jun protein in thec-jun-transfected clones is driven by the metallothioneinpromoter.
The expression of c-los was also examined at the mRNAand protein levels in c-los-transfected clones; all of the six
transfectants examined exhibited increased expression ofc-los mRNA and protein (Figs. 28 and 38). In the presenceof CdCI,, an increase in c-los was observed (Fig. 3B).However, the increased expression of c-los in these clonesdid not result in spontaneous differentiation whether cellswere grown in the presence (Table 1 ) or the absence (datanot shown) of serum. Thus, in contrast to c-jun, expressionof c-los was not sufficient to initiate the maturation ofWEHI-3B D� cells, although both are components of theAP-i transcription factor.
To confirm that the expression of c-jun can result in theinitiation of the differentiation of WEHI-3B D� cells andthat possible differentiation factors present in the serumwere not responsible for the entrance of cells into a differ-entiation pathway, the six clones were grown in serum-freemedium in the presence or absence of CdCI2, and their
abilities to reduce NBT were measured. As shown in Table2, all but clone D*SFJ3 exhibited a higher degree of mat-uration in the presence of CdCI,. Clone D�SFJ3, which hadlittle or no increase in the expression of c-Jun protein in thepresence of CdCI2 (Fig. 3), did not exhibit a significantincrease in the percentage of NBT-positive cells in the
A
Origin -
MW(Kd)
97.4 -
21.5 -
14.4 -
B
Clone DSFVI
CdCI2 - 4
Origin -
MW(Kd)
97.4 -
66.2
45.0 -
D4FI D’F2 D�F3 D’F4
4 - + - 4 - 4
I 2 3 4 5 6 7 8 9 10 11 12 13 14
D’FS DF6
+ - 4
. � �-r----- -�- -L��
�:-. � .�..
� �
�
) c-Fos
�-�--�
(elI Growth & Differentiation 747
Clone D�SFV1
CdCI2 - 4
I 2
66.2
D’SFJI DSFJ2 DSFJ3 DSFJ4 DSFJ5 D�SFJ6
- 4 - 4 - 4 - 4 - 4 . 4
3 4 5 6 7 8 9 10 ii 12 13 14
45.0 -
---�-� -� �-.
Fig,’. I. Expression of ( -Ion andc-Fos proteins in IMLI 1 t - and
075/1 5-translected WEHI-Ill D’(dl Is. [he lransfeclants weretreat(’dl exactly as ii) Fig. 2� Total((‘I lul,ir extracts ssere StiI)jecte(l
to SeI).lr.itiOn 1)’, SDS-polyacryl-arlli(k’ gel electrophoresis and
then to Western blotting with anti-
c-Ion ,od anti-c-Fos .intil)0(lieS. A,
(-1(11) (‘xI)r(’ssiotl it-i I)MTI 1 1-trans-le tt’(l ( lones; B. ( -Fos expressionin o7 5/1 5-transfecteol ccl Is. The
lane .issignnients are the same asin(lic,it(’(l in the legend to Fig. 2.
31.0 -
31.0 -
21.5 -
14.4 -
�-. ��-. ‘� � --. � � � c-Jun
-.-- D+SFV1
-..-- D+SFJ5
20
0 10 20 30
748 WEHI-3B D� Cell Differentiation by c-jun
Table 2 Differentiation of WEHI-3B D� leukemia cells transfected withthe c-jun-containing construct pMTJi 1 in serum-free medium
NBT-positive cells (%)Constructs Clone
No cadmium Cadmium”
D�SFV 1 3 6
D�SFJ 1 33 73
2 6 13
3 6 4
4 27 42
5 40 74
6 18 30
a WEHI-3B D� cells were transfected with pMTJ1 1 , selected with G-41 8,
cloned in modified McCoy’s 5A medium containing 1 5% FBS, and expandedin X-VIVO-1 5 serum-free medium. D�SFV and D�SFJ are transfectants ofvector alone and pMTJ1 1 , respectively.
b Cells were treated with 1 0 �M CdCI2 in �-VIVO-1 5 medium for 3 days.
(IUV
00�
I-
z
Cadmium Chloride (jiM)
Fig. 4. The effects of various concentrations of CdCl2 on the differentiationof c-Jun-transfected D�SFJ5 cells in serum-free medium. D�SFV1 vector-
transfected cells and D�SFJ5 c-jun-transfected cells were cultivated in X-VIVO-1 5 serum-freemedium supplemented with different concentrations of
CdCI2 for 3 days. Differentiation was then determined by the percentage ofNBT-positive cells.
presence of cadmium. Fig. 4 shows the effects of variousconcentrations of CdCl2 on the differentiation of D�SFJ5,one of the c-jun transfected clones, in serum-free medium.D�SFVi , a vecton-transfected clone, treated in exactly thesame manner did not respond to any of the concentrationsof CdCl2 used by entering a differentiation pathway. Incontrast, the c-jun-tnansfected clone, D�SFJ5, exhibited aCdCl2 concentration-dependent degree of differentiation.These results strongly suggest that expression of c-jun itselfis sufficient to initiate the maturation of WEHI-3B D” cells.
In serum-containing medium, many freshly establishedc-jun clones exhibited a mature phenotype spontaneously,with a high percentage of cells being NBT-positive. How-even, the degree of spontaneous differentiation of the c-junclones (i.e., the percentage of NBT-positive cells in theabsence of CdCI2) decreased gradually with time. Thus,after i-3 weeks, these clones were virtually indistinguish-
Table 3 Effects of the constitutive expression of c-jun on retinoic acid-induced differentiation of WEHI-3B D� cells’
TreatmentConcentration
(pM)
NBT positivity
WEHI-3B D� WD�J32
None 1 1
CdCI2 80 1 6
Retinoicacid 0.1
0.5
1
35
7
13
i4
23
3038
69
30
58
62
6476
82
a Cells were treated with CdCl2 or retinoic acid in modified McCoy’s 5Amedium containing 1 5% FBS for 3 days, and differentiation was determined
by NBT positivity.
able from parental cells (data not shown). These clones mayeither represent cells which have lost the capacity to ex-press the plasmid c-jun on represent cells which wereblocked in their ability to differentiate. Examination of thelevels of c-jun expression in these clones at this stage byNorthern hybridization and Western blotting indicated thatthe cells were still expressing high levels of c-jun mRNAand protein (data not shown). To determine whether thesecells were still capable of undergoing differentiation whilecontinuously expressing c-jun, clone WD�J32 was exposedto different concentrations of netinoic acid, and the resultsare shown in Table 3. CdCl2 did not cause significantdifferentiation under the conditions used. However, c-jun-transfected cells were considerably more sensitive to reti-noic acid than WEHI-3B D� parental cells used as a con-trol. Thus, 0.5 �M netinoic acid produced 58% NBTpositivity in WD�J32 cells; to achieve this degree of differ-entiation, parental WEHI-3B D� cells required between
40 � �M netinoic acid.The enhancement of the capacity of netinoic acid to
induce the differentiation of WEHI-3B D� cells by theectopic expression of c-jun was also observed in freshlyestablished clones after transfection (Fig. 5). Thirty-threec-jun and 34 c-los clones were treated with 50 �M CdCI2
and 7 �M netinoic acid for 3 days, and the percentage ofNBT-positive cells was determined; a positive regulatoryeffect was observed in c-jun-containing clones but not inc-los-tnansfected clones. Thus, under these conditions, 74%
of parental WEHI-3B D� cells were NBT positive, while 25of 33 c-jun-transfected clones exhibited greater than 74%differentiation, with many of the clones having more than9O% of the cells in the mature state. In contrast, more thanone-half of the c-los-containing clones (1 8 of 34) had alower percentage of differentiated cells than the parentalcontrol. Furthermore, the average percentage of diffenenti-ated cells in c-jun clones was 20% higher than that in c-losclones. These findings suggest that c-jun may be involved inthe retinoic acid signal transduction pathway that leads tothe induction of the mature state induced by the netinoid.
Discussion
Cell growth and differentiation are reciprocally relatedprocesses. Thus, when cells are undergoing differentiation,growth is decreased and eventually terminated when cellsreach a mature state with a programmed, finite life span.The use of gene transfer techniques to investigate the pos-
A
14
12
10
II
6
4
�1
II 1(1 20 3(1 40 So 60 70 SO 90
.�
zV
C
U
4
12
.� t(
C
zV
C
U
NB1’ Positivity (%)
B
S
4
0 10 20 31) 411 50 60 70 5(1 �)t) 10(1
NB1’ Positivity (%)
Fig. 5. Differential effects of c-jun and c-los transfection of WEHI-3B Dcells on differentiation induced by retinoic acid. Following transfections, 33
c-jun clones (A) and 34 o -los clones (B) were treated with 7 �M retinoic acidand 50 psi CdCI in modified M Coy’s 5A medium containing t 5/. FBS for
3 days. Ditterentiation was assayed by NBT reduction, and the clone num-hers are plotteol against the percentage of NBT-positive cells.
Cell Growth & Differentiation 749
sible involvement of genes in the control of proliferationand differentiation can be a problem with genes that arecapable of initiating the differentiation process, particularlyif cells expressing that gene undergo terminal maturationand thereby are eliminated because of their inability toduplicate during the period of antibiotic selection of trans-fected clones. In such instances, the clones that are ob-tamed may not provide correct information about the func-tion of the gene that is introduced. To avoid this possibility,we used the inducible metallothionein promoter in a plas-mid that also contained a portion of the polyoma largeT-antigen coding sequence that has been shown to pro-duce immortalization of murine fibroblasts (28) in aneffort to retain cells that express a mature phenotype. Astriking difference was found in the proportion of differ-entiating cells that occurred, with measurements as earlyafter antibiotic selection and expansion of single cellclones as possible (stage 1). Under these conditions, ahigh degree of spontaneous differentiation coupled withslow growth was observed, even in the absence of CdCI,.The “spontaneously” differentiated cells in the popula-tion declined, and the growth rate increased with time
(stage 2), with no difference being observed betweenc-jun-transfected cells and parental cells. The shift in theculture to an undifferentiated phenotype was not due to
an inability to express ectopic c-jun, as indicated byNorthern hybridization and Western blot measurements.A basal level of expression of c-jun occurred, presumablydue to the leakiness of the metallothionein promoter, a
phenomenon that resulted in the “spontaneous” differen-tiation of transfected cells; in these clones, cellulargrowth declined, and most of the population exhibited adifferentiated phenotype. With time, the mature cells thatcould not replicate were replaced by cells that wereresistant to the initiation of differentiation caused by theectopic expression of c-jun. Thus, no further “spon-taneous” differentiation occurred at stage 2. Whenc-jun-expressing cells were switched to serum-freemedium immediately after the establishment of clones,the clones could be maintained for a longer period oftime at stage 1 . A two-stage phenomenon of differentia-tion was also observed in FDC-Pmix hematopoietic stemcells by the ectopic expression of the transfected granu-locyte-macrophage colony-stimulating factor gene (29).
c-jun-transfected cells at stage 2 continued to expresshigh levels of c-jun and retained the capacity to enter adifferentiation pathway when treated with retinoic acid.Since retinoid treated c-jun-expressing clones exhibited agreater sensitivity to the maturation-producing activity ofretinoic acid, it suggests that c-jun retains the capacity tohave a positive regulatory role in cellular maturation andthat the ectopic expression of c-jun at stage 2 is not suffi-cient to initiate maturation. This implies that another eventoccurs in the c-jun-transfected cells that results in the inhi-bition of the terminal differentiation-initiating ability ofc-jun, insuring the survival of the cellular population. Themechanism involved in this phenomenon is unknown. Theinhibition may occur at a step in the c-jun signal pathway ordirectly at the level of the c-Jun protein through the repres-sion of the function of c-Jun by any member of the jun or losfamilies, or by some unidentified inhibitor. Nakabeppu andNathans (30) have reported that a naturally occurring trun-cated form of FosB (z�FosB) inhibits gene activation by Junor by the Fos/Jun complex, presumably by competing withfull-length Fos protein at the levels of dimenization with Junand the binding of the heterodimer to DNA. Other possibleinhibitors of the function of c-Jun have been described.Thus, using an in vivo competition assay, Baichwal andTijan (31) have reported the existence of a cell-specificinhibitor of c-Jun; a dominant inhibitor of Fos/Jun, namedlP-i, whose activity is modulated by phosphorylation, hasbeen identified in several different cell types (32), andJun-B, a member of the Jun family, has been shown to be anegative regulator of c-Jun (33, 34). We are currently in-vestigating the mechanism by which the initiation of thedifferentiation of WEHI-3B D� cells by the expression ofc-jun is inhibited at stage 2.
Treatment of several leukemia cells lines, including HL-60, U937, and WEHI-3B D�, with TPA produces macroph-agic differentiation accompanied by an early and transientincrease in c-los protooncogene mRNA (3, 35-37), indicat-ing that c-los expression may be involved in macrophagicdifferentiation. However, c-los expression is not sufficientnor obligatory for macrophagic maturation of HL-60 orU937 cells (38, 39). In agreement with this finding, trans-fection of WEHI-3B D� cells with c-los did not result in theinitiation of differentiation, even though c-los expression
750 WEHI-3B D� Cell Differentiation by c-jun
was increased by exposure to CdCI2 (40). While this latterfinding confirmed earlier observations that c-los expressionis not sufficient to initiate macnophagic differentiation, mi-tiation of the expression of c-los by treatment of WEHI-3BD� cells with cadmium resulted in a 50% decrease in thedegree of granulocytic maturation induced by retinoic acidon by aclacinomycin A (40). In an analogous approach,introduction and stimulation of the c-los gene in F9 tenato-carcinoma cells produced morphologically altered colonies(41). Thus, it appears that sufficient evidence does notexist at this time to exclude the possibility that the c-losgene has a role in the regulation of cellular proliferationand differentiation.
Materials and Methods
Structures of Plasmids. The c-los expression plasmidp75/i 5 was obtained from Dr. RoIf MUller, EuropeanMolecular Biology Laboratory, Heidelberg, Federal Repub-lic of Germany (4i ). To construct a c-jun expression plas-mid, p75/i 5 was digested with BamHI to remove the c-losDNA and neligated to generate the vector p75/i 5V for c-jun
subcloning. The vector contains: the bacterial neo genewith an SV4O promoter, which confers resistance to theantibiotic G-4i 8 in eukanyotic cells; the human metallo-thionein promoter, under which the expression of theinserted gene is stimulated by treatment of cells withcadmium; and part of the polyoma virus early regionsequence, including the origin of replication, the pro-moten, the enhancer, and 40% of the amino terminal ofthe large T-antigen coding sequence, which has beenshown to be sufficient for immortalization of muninefibroblasts (28). The rat c-jun complementary DNA, agift from Dr. Frank Rauscher, Ill, of the Wistar Institute,Philadelphia, PA, was subcloned into the EcoRi site ofpSP72. The c-jun DNA was then removed by BamHI-BglIl digestion and inserted into the BamHl site of p75/1 5V to generate pMTJi i.
Cell Culture and Differentiation. WEHI-3B D� cellswere grown in suspension culture in McCoy’s 5A modifiedmedium with 15% FBS (GIBCO, Grand Island, NY) or inserum-free medium X-VIVO-i 5 (BioWhittaken, Walkers-ville, MD) at 37#{176}Cin a humidified atmosphere of 95% airand 5% CO2. Cells were seeded at a density of 5 x 10�cells/mI in culture medium and maintained in exponentialgrowth by subpassage every 2 days. Cell numbers weredetermined using a Model ZM particle counter connectedto a model 256 Channelyzer (Coulter Electronics, Inc.,Hialeah, FL).
Exponentially growing cells (5 X i04 cells/mI) were ex-posed to an inducer of differentiation or to vehicle in freshculture medium to initiate maturation. A stock solution ofall-trans-netinoic acid (Sigma Chemical Co., St. Louis, MO)was prepared and used as a differentiation inducer asdescribed previously (25). Cadmium chloride (SigmaChemical Co.) was used to stimulate the expression of thegenes under the control of the metallothionein promoter.
The ability of cells to reduce NBT was used as a func-tional marker of differentiation. Cells (106 cells/mI) wereincubated in serum-free McCoy’s SA medium containing0.i % NBT and 2 �M TPA at 37#{176}Cfor 30 mm with shaking.The percentage of NBT-positive cells with blue-blackfonmazan deposits was determined microscopically on 200consecutive cells.
The level of Mac-i (CD1 1 b) expression on the surface ofcells was measured by immunofluorescent staining andflow cytometry. Cells (1 06) were washed once with PBS andthen incubated with 100 p1 of FITC-conjugated anti-Mac-imonoclonal antibody at 10 pg/mI in buffer (PBS containing0.1% bovine serum albumin) on ice for 30 mm. After cellswere washed twice, the amount of antibody bound to thecell surface was analyzed using a FACS IV flow cytometen.Excitation of the fluorochnome was achieved at 488 nmwith a Spectra-Physics 2025 argon ion laser (Spectra-Phys-ics, Mountain View, CA). The FITC emission was collectedthrough a 530/30 nanometer land pass filter (Omega Opti-cal, Brattlebono, VT). Twenty thousand cells were observedfor each sample.
Morphological changes indicative of maturation wereassessed following staining with a modified Wright stainusing Diff-Quick stain set (American Scientific Products,McGaw Park, IL). Two X 10� cells were subjected to acytospin (Shandon Southern Centrifuge, Sewickley, PA) at800 rpm for 5 mm. The cytospin preparations were stainedaccording to the instructions of the supplier and examinedby light microscopy.
Transfedion and Single Cell Cloning. Exponentiallygrowing WEHI-3B D� cells were washed with PBS andresuspended in PBS at 1 x i 0� cells/mI. The cell suspension(0.8 ml) was incubated with i 5 pg of linearized DNA for i 0mm. Cells were then transfected by electroponation using aBio-Rad Gene Pulsen apparatus at 625 V/cm, 960 pF. Tenmm later, cells were grown in 10 ml of modified McCoy’s5A medium containing 15% FBS. G-4i8 was added at aconcentration of 400 pg/mI following overnight growth.After a 2-week selection period with G-4i 8, transfectedcells were single-cell cloned using a flow cytometen whichplaced one cell in each well of a 96-well plate. Each clonewas expanded in multiwell plates.
RNA Isolation, Northern Blotting, and Hybridization.Total cellular RNA was isolated from 2 x 106 cells using theguanidmne isothiocyanate/acid/phenol method described byChomczynski and Sacchi (42), separated by 1% aganoseformaldehyde gel electrophonesis, and transferred ontoGeneScreen membranes (DuPont NEN Research Products,Boston, MA) using a vacuum blotter. The membranes wereprehybnidized in Rapid-hyb buffer (Amensham Corporation,Arlington Heights, IL) for 45 mm at 65#{176}C,hybridizedwith an [a-32PIdCTP random primer-labeled c-jun probeat a concentration of 1 06 cpm/ml for 3 h, washed with 2XSSC (1 X SSC = 0.1 5 M NaCl-0.0i 5 M sodium citrate),0.1% SDS at room temperature for 45 mm; 0.5X SSC,0.1% SDS at 65#{176}Cfor 15 mm; and 0.1X SSC, 0.1% SDSat 65#{176}Cfor 1 5 minutes. The c-jun mRNA was visualizedby autoradiography.
Western Blotting. Two X 10� cells were collected bycentnifugation and resuspended in 200 p1 of PBS containinga mixture of protease inhibitors (2 mt�i phenylmethylsulfonylfluoride, 2 ms�i ethylenediaminetetnaacetic acid, 2 mtsi 0-phenanthroline, 1 pg/mI of leupeptin, 1 pg/mI of aprotinin,
and i pg/mI of pepstatin A), lysed by adding an equalvolume of 2X loading buffer (0.1 25 M Tnis-HCI, pH 6.8-4%SDS-20% glycerol-5% mencaptoethanol), and sonicated tobreak genomic DNA. After boiling for 5 mm, extracts wereseparated by electrophonesis on a 7.5-15% gradient SDS-polyacrylamide gel and transferred onto nitnocellulosemembranes. The membranes were blocked with i O% dry
milk in TBST (20 mM Tnis-HCI, pH 7.6-1 37 msi NaCI-0.i %Tween-20) for 1 h, rinsed twice with TBST-M (TBST con-
Cell Growth & Differentiation 751
taming 5% dry milk) for 10 mm, incubated with rabbitpolyclonal anti-c-Jun antibody (Oncogene Science, Inc.,Manhasset, NY; 1 :400 diluted in TBST-M) for 1 h, washed
with four changes of TBST-M for a total of 1 h, and thenincubated with horseradish penoxidase-conjugated donkey
anti-rabbit lgG for 1 h and washed for 2 h with TBST-M (sixchanges). The immunoneactive proteins were visualized bythe en hanced chemilum inescence method (AmenshamCorp.).
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