Download Max and inhibitory c-Myc mutants induce erythroid di

Oncogene (1997) 14, 1315 ± 1327
 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00
Max and inhibitory c-Myc mutants induce erythroid di€erentiation and
resistance to apoptosis in human myeloid leukemia cells
Matilde CanÄelles1, M Dolores Delgado1, Kathy M Hyland2, Ana Lerga1, Carlos Richard3,
Chi V Dang2 and Javier LeoÂn1
1
Departamento de BiologõÂa Molecular, Facultad de Medicina, 39011 Santander, Spain; 2Division of Hematology, Department of
Medicine, The John Hopkins University School of Medicine, Baltimore, Maryland 21205; 3Servicio de HematologõÂa, Hospital
Universitario MarqueÂs de Valdecilla, 39011 Santander, Spain
We have used the human leukemia cell line K562 as a
model to study the role of c-myc in di€erentiation and
apoptosis. We have generated stable transfectants of
K562 constitutively expressing two c-Myc inhibitory
mutants: D106-143, that carries a deletion in the
transactivation domain of the protein, and In373, that
carries an insertion in the DNA-interacting region. We
show here that In373 is able to compete with c-Myc for
Max binding and to inhibit the transformation activity of
c-Myc. K562 cells can di€erentiate towards erythroid or
myelomonocytic lineages. K562 transfected with c-myc
mutants showed a higher expression of erythroid
di€erentiation markers, without any detectable e€ects
in the myelomonocytic di€erentiation. We also transfected K562 cells with a zinc-inducible max gene. Ectopic
Max overexpression resulted in an increased erythroid
di€erentiation, thus reproducing the e€ects of c-myc
inhibitory mutants. We also studied the role of c-myc
mutants and max in apoptosis of K562 induced by
okadaic acid, a protein phosphatases inhibitor. The
expression of D106-143 and In373 c-myc mutants and
the overexpression of max reduced the apoptosis
mediated by okadaic acid. The common biochemical
activity of D106-143 and In373 is to bind Max and
hence to titrate out c-Myc to form non-functional Myc/
Max dimers. Similarly, Max overexpression would
decrease the relative levels of c-Myc/Max with respect
to Max/Max. The results support a model where a
threshold of functional c-Myc/Max is required to
maintain K562 cells in an undi€erentiated state and to
undergo drug-mediated apoptosis.
Keywords: Max; c-Myc mutants; K562; erythroid
di€erentiation; apoptosis; okadaic acid
Introduction
c-Myc contains a transcriptional activation domain
and a basic/helix ± loop ± helix/leucine zipper (bHLHLZ) domain that mediates sequence-speci®c DNA
binding and heterodimerization with Max, itself
another bHLH-LZ protein. All the known biological
functions of c-Myc depend on its dimerization with
Max (reviewed in Amati and Land, 1994; Vastrik et al.,
1994). Myc/Max heterodimer binds to a DNA
consensus sequence CACGTG, termed E Box Myc
Correspondence: J LeoÂn
Received 18 July 1996; revised 8 November 1996; accepted 11
November 1996
site (Ems) (Blackwood and Eisenman, 1991; Prendergast et al., 1991). Myc/Max activates transcription of
reporter genes carrying Ems in their promoters, while
the homodimer Max/Max is inactive as a transcriptional activator (Kretzner et al., 1992; Kato et al.,
1992; Amin et al., 1993; Gu et al., 1993). Also, the
overexpression of Max results in suppression of cell
transformation mediated by c-Myc (Makela et al.,
1992; Prendergast et al., 1991; Mukherjee et al., 1992;
Amati et al., 1993a; Gu et al., 1993; Koskinen et al.,
1994; Lindeman et al., 1995). These data are consistent
with the idea that inactive Max/Max dimers compete in
vivo with Myc/Max for common DNA binding sites.
Expression of c-myc is induced during mitogenic
stimulus and required for cell growth, while terminal
di€erentiation of many cell types is accompanied by
down-regulation of c-myc expression (Blackwood et al.,
1992; Marcu et al., 1992; Kato and Dang, 1992;
Meichle et al., 1992; Evan and Littlewood, 1993).
Myeloid leukemia cell lines have been broadly used as
models to study the molecular basis of the proliferation-di€erentiation switch. Following induction of
di€erentiation in myeloid cell lines, c-myc is downregulated. This has been found in murine (M1, MEL,
WEHI3B) and human cell lines (HL60, U937)
(reviewed in Marcu et al., 1992). Consistently,
constitutive expression of c-myc inhibits the chemically-induced di€erentiation of some of these cell lines
(Chisholm et al., 1992; Coppola and Cole, 1986;
Dmitrovski et al., 1986; Larsson et al., 1988) and
inhibition of c-myc expression induce di€erentiation
(Holt et al., 1988; Prochownik et al., 1988; Nguyen et
al., 1995). Paradoxically, it has been shown that c-Myc
overexpression induces apoptosis in di€erent cell
systems under conditions where c-Myc is normally
down-regulated (reviewed in Harrington et al., 1994;
Packham and Cleveland, 1995). The apoptosis-promoting activity of c-myc was originally observed in rodent
®broblasts but also takes place in murine myeloid cells
deprived of IL3 or exposed to antiproliferative
cytokines (Askew et al., 1991; Lotem and Sachs,
1993; Selvakumaran et al., 1994).
Human K562 is a bipotential cell line that expresses
erythroid markers and can be further di€erentiated in
vitro towards erythroid lineage with 1-a-D-arabinofuranosylcytosine (ara-C) and other inducers (Rowley et
al., 1981), or towards myelomonocytic lineage (with
expression of monocytic and megakaryocytic markers)
with phorbol esters (Kamano et al., 1990; Shen et al.,
1992). We have previously shown that c-myc is downregulated during the erythroid and myelomonocytic
di€erentiation of K562 cells (Gomez-Casares et al.,
Max and Myc in differentiation and apoptosis of K562 cells
M CanÄelles et al
1316
1993) and that c-myc overexpression leads to a partial
inhibition of erythroid di€erentiation in K562 cells
(Delgado et al., 1995). On the other hand, although
K562 cells are relatively resistant to apoptosis the
protein phosphatases inhibitor okadaic acid (OA)
readily induces apoptosis in K562 (Zheng et al., 1994;
Lerga et al., 1995). Therefore K562 cell line provides a
useful model system to study the involvement of c-Myc
in growth, di€erentiation and apoptosis. In the present
work we have investigated the e€ects of dominantnegative c-myc mutants and max on di€erentiation and
apoptosis of K562. Two mutant c-myc genes were
used: D106-143 and In373. These mutants were chosen
because: (i) they are completely inactive for Rat-1A
cells transformation and rat embryo cells (REC) MycRas cotransformation (Stone et al., 1987); (ii) they are
two of the most potent dominant negative c-myc
mutants inhibiting the transformation of Rat-1 cells
by v-ABL or BCR/ABL, and in the case of D106-143,
inhibiting the cotransformation of REC by Myc-Ras
(Dang et al., 1989; Sawyers et al., 1992), and (iii) they
carry alterations in two di€erent domains of c-Myc
protein: the transactivation domain and the speci®c
DNA binding domain. The Max-dimerizing region is
not a€ected in the mutants used. D106-143 lacks
amino acids 106 to 143 of wild-type c-Myc. This region
is able to act as an independent transcriptional
activation domain and is responsible for the transformation and transrepression activities of c-Myc (Kato et
al., 1990; Li et al., 1994; Brough et al., 1995; Lee et al.,
1996). The Myc mutant In373 carries an insertion in
the DNA binding region (Stone et al., 1987) and we
show now that it binds to Max, but the dimer is unable
to bind DNA. We present evidence suggesting that the
expression of either c-myc inhibitory mutants or max
enhance the erythroid di€erentiation of K562 cells
without any detectable e€ect on myelomonocytic
di€erentiation. Furthermore, we have found that
expression of max and c-myc mutants signi®cantly
reduces the drug-mediated apoptosis of K562.
Results
Characterization of the In373 mutant
To study c-Myc role in di€erentiation of K562 cells, we
set out to obtain cell lines constitutively expressing cmyc inhibitory mutants. We used for transfections two
potent dominant negative c-myc mutants: D106-143 and
In373. As the biochemical activities of In373 are
unknown, we ®rst analysed its ability to bind DNA,
dimerize with Max and inhibit c-Myc cotransformation
activity. Previous studies have demonstrated that c-Myc
alone is unable to bind the E-box sequence, CACGTG
unless at very high concentrations. However, a
truncated form of c-Myc (tMyc), which contains amino
acids 342 ± 439, is able to bind the E-box sequence as a
homodimer (Kato et al., 1992). When mixed with Max
protein, tMyc is able to form heterodimers with Max
that bind DNA resulting in three resolvable proteinDNA complexes: tMyc-tMyc, Max-Max and tMycMax. To determine if the dominant negative tIn373
protein, which also contains Myc amino acids 343 ± 439
with an insertion of four serins at residue 373, is able to
a€ect the DNA binding properties of these complexes,
we have produced and puri®ed these hexahistidine
tagged proteins from bacteria (Figure 1a). Although
In373 contains four extra amino acids, the polylinker
sequence of pDS-Myc(342 ± 439) contributes to amino
acids that exceed the In373 polypeptide by four amino
acids. Hence the In373 polypeptide showed a faster
mobility on SDS ± PAGE.
In373 is unable to bind DNA (Figure 1b, lane 4),
whereas tMyc forms a distinct homodimeric complex
(Figure 1b, lane 3). In this experimental design, Max
(6 ng) was mixed with an excess of tMyc (600 ng) so that
tMyc-tMyc homodimers and tMyc-Max heterodimers
are readily detected on EMSA (Figure 1b, lane 5). The
Max-Max homodimer existed at low levels under these
conditions. Addition of increasing amounts In373
(Figure 1b, lanes 6 to 9) resulted in diminishing intensity
of shifted bands corresponding to tMyc/Max, Max/Max
and tMyc/tMyc. These results indicate that recombinant
In373 protein is able to disrupt DNA binding by tMyc
and Max homodimers and heterodimers.
To determine whether In373 is able to heterodimerize with Max in vivo, we tested the abilities of these
proteins to interact using a mammalian two-hybrid
assay that had been reported previously (Dang et al.,
1991). The dimerization domains of wild-type c-Myc or
of In373 (including the insertion mutation) were fused
to the Ga14 DNA binding domains. Constructs
encoding these proteins were cotransfected with either
the control pNLVP plasmid, which encode the VP16
activation domain, or with pVPMax that encodes Max
fused to the VP16 activation domain. The interaction
of these hybrid proteins would reconstitute the
function of GAL4 to transactivate a GAL4 driven
reporter, G5-E1B-CAT (Figure 1c). When compared to
the controls with pNLVP, both wild-type c-Myc and
In373 sequences were able to interact with Max in
transfected cells. The protein levels of GAL4-Myc and
GAL4-In373 are comparable in transfected CHO cells
as determined by immunoblotting (data not shown).
These results indicate that the insertion at position 373
did not disrupt Myc ability to interact with Max.
Although the In373 mutant was shown to be nontransforming (Stone et al., 1987) and to interrupt
oncogenic Abl-mediated transformation (Sawyers et
al., 1992), it is unknown whether it is able to inhibit cMyc transforming activity. Therefore we analysed its
e€ect on a cotransformation assay of REC mediated
by ras and myc. Similar to the D106-143 mutant (Dang
et al., 1989), In373 is able to dramatically inhibit
transformation of REC by wild-type c-Myc and
activated Ras. As a control, we also cotransfected
In6, another insertional mutant which does not act as
an inhibitory mutant (Stone et al., 1987). Cotransfection of In6 increased the number of foci in the presence
of wild-type c-Myc and activated Ras (Figure 1d). This
was the expected result as In6 is able to cotransform
REC. Together with previous results in stably
transfected cells (Sawyers et al., 1992), our results
indicate that In373 is a potent dominant negative
inhibitor of c-Myc transforming activity.
Generation of K562 cell lines stably transfected with cMyc mutants
The mutants D106-143 and In373 were transfected into
K562 cells by electroporation and several G418-
Max and Myc in differentiation and apoptosis of K562 cells
M CanÄelles et al
1317
a
b
In373
–
1
–
2
–
3
+
4
–
5
6
7
8
9
+
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+
11
–
12
–
–
+
+
+
+
+
+
+
+
+
+
–
+
+
–
–
–
Std tMyc In373 Max
50
33
Max-Max —
tMyc-Max —
22
14
tMyc-tMyc —
free
—
probe
Max
tMyc
–
–
c
+
–
–
+
d
Figure 1 Characterization of In373. (a) Puri®ed recombinant hexahistidine fusion protein of tMyc, In373 or Max were resolved on
SDS ± PAGE and stained with Coomassie blue. The left lane (Std) shows prestained standards and the molecular masses (kDa) are
indicated on the left margin. For Myc and In373, a contaminating 30 kDa protein is noted. (b) The Myc mutant In373 inhibits
DNA binding by tMyc and Max in electrophoretic mobility shift assay. The probe is a 260 bp CACGTG containing oligonucleotide
(pDW14) that resolves the di€erent DNA bound dimers (indicated at the left margin). The amounts of recombinant proteins in each
reaction were: Max, 6 ng; tMyc, 0.6 mg; tIn373, 0.6 to 3.3 mg (lanes 5 to 9). (c) Mammalian two-hybrid assay demonstrates an
interaction between Max and either wild-type Myc or In373. Gal-Myc is the GAL4 DNA binding domain fused to c-Myc amino
acids 262 ± 439, Gal-In373 contains c-Myc amino acids 262 ± 439 with an insertion of four serines at residue 373. pNLVP encodes
the VP16 transactivation domain. VP-Max is Max fused at its N-terminus to the VP16 transactivation domain. The CAT activities
were derived from duplicated experiments and normalized to the controls with pNLVP. (d) In373 inhibits Myc cotransformation of
rat embryo cells in the presence of activated H-Ras (EJ-ras). Each 100 mm plate (in quadruplicates) of rat embryo cells were
lipofected with 5 mg of Ras plasmid alone or with 5 mg of Myc expression plasmid. Cotransfections were with 5 mg of either In373
or In6 genes. Transformed foci per plate are indicated on the ordinate
resistant clones were selected. In order to assess the
stable integration of the transfected genes into the cell
genome, three K562 sublines transfected with D106143 (KD2, KD4 and KD11) and two transfected with
In373 (KIA and KIB) were subjected to Southern
analysis. The results (Figure 2a) indicate that the
transfected c-myc genes are integrated in the genome of
the ®ve analysed clones. The hybridization revealed the
presence of multiple copies of the c-myc EcoRI
fragment and of extrabands for the ®ve analysed
clones. The intensity of the band of the transfectants as
compared with the corresponding of parental cells
indicates that transfected cells carried multiple copies
of the c-myc gene. Northern blotting analysis showed
in all ®ve cases an augmented expression of steadystate c-myc mRNA with respect to non-transfected
K562 cells (Figure 2b). The increase ranged from threeto 10-fold, as assessed by ®lm densitometry (not
Max and Myc in differentiation and apoptosis of K562 cells
M CanÄelles et al
1318
shown). The K562 clones transfected with D106-143
(KD2, KD4 and KD11) were analysed for mutant Myc
protein expression by Western blotting, and for all the
cases a c-Myc doublet was observed, corresponding to
the endogenous and exogenous c-Myc proteins (Figure
2c). As reported (Stone et al., 1987), the mobility of
D106-143 protein was smaller than that of the
endogenous protein. In sharp contrast with the
mRNA expression, the levels of D106-143 protein in
the transfected cells were 4 ± 6-fold lower (by ®lm
densitometry) than those of the wild-type c-Myc. No
information could be obtained on the amount of In373
in the transfectants as the mutant protein has about the
a
K562 KD2
KD4
KD11 KIA
KIB
M
— 21,2
— 5,1
— 4,2
— 3,5
—2
b
K562
KD2
KD4
KD11
KIA
KIB
c-myc
rRNAs
c
K562
KD2
KD4
KD11
M
— 97
▼ ▼
Myc
D106-143
— 50
Figure 2 Analysis of K562 cell lines stably transfected with cmyc mutants. (a) Presence of c-myc exogenous sequences in K562
sublines tranfected with D106-143 (KD2, KD4 and KD11) and
with In373 (KIA, KIB). High-molecular weight DNAs from the
cell lines were subjected to Southern analysis as described in the
text, and hybridized to a human c-myc probe. The size of markers
in kb are indicated at the right. (b) c-myc mRNA expression in
transfected K562 cell lines. Total RNAs were prepared from the
indicated cell lines and analysed by Northern analysis using a
human c-myc probe. The lower panel shows the ®lter stained with
ethidium bromide to assess RNA integrity and loading. (c)
Immunoblot of c-Myc proteins in K562 and KD2, KD4 and
KD11 cell lines. The position of endogenous normal c-Myc and
D106-143 mutant are indicated. The position of the molecular
weight markers (kDa) are indicated at the right
same size that the endogenous c-Myc. To be used as
controls, several K562 lines transfected with a plasmid
carrying a G418 resistance gene were generated
(Kneo2, Kneo3, Kneo5, Kneo6, Kneo8).
c-Myc mutants enhance erythroid di€erentiation of K562
Growth of K562 cells transfected with mutant c-myc
genes was reduced by about 25% with respect to
parental cells and vector-transfected cells, as assessed
by [3H]thymidine incorporation and cell counts. Also,
both mutants drastically reduced K562 clonogenicity in
agar after transfection (not shown). In contrast to the
cells expressing mutant c-myc genes, cells overexpressing wild-type c-myc (KmycJ cells in the presence of
zinc) or Kneo cells did not show signi®cant di€erences
in their growth rate with respect to uninduced or
parental cells (not shown). We studied the e€ects of cMyc mutants in myelomonocytic and erythroid
di€erentiation. Transfectant cells did not show any
increase of myelomonocytic/megakaryocytic markers as
expression of GpIIb ± IIIa, cluster formation, adherence to plastic and fraction of cells positive for the
nitroblue tetrazolium reduction test. Furthermore,
when myelomonocytic di€erentiation was induced by
treatment with 10 nM TPA for 3 days, the extent of
di€erentiation of KD2, KD4, KIA and KIB cell lines
was similar to that of parental K562 cells or Kneo cell
lines, as assayed by cluster formation and nitroblue
tetrazolium reduction (about 30% of positive cells in
all lines) (not shown). Therefore, myelomonocytic
di€erentiation of K562 seemed to be una€ected by
expression of Myc mutants.
We then analysed the erythroid di€erentiation in
K562 cells expressing inhibitory c-myc genes, as
compared with the parental cells or neo expressing
cells. Cell lines KD2, KD4, KIA and KIB showed a
signi®cant increase in the erythroid di€erentiation as
assessed by the expression of e-globin mRNA, an
embryonic globin expressed in K562 (Charnay and
Maniatis, 1983) (Figure 3a). The amounts of globin
mRNAs are compared in Figure 3b to show that the
basal expression of e-globin is 2 ± 3-fold higher than in
parental cells. This result was con®rmed by the
cytochemical reaction of benzidine, which detects
hemoglobinized cells. The fraction of benzidinepositive cells in KD2, KD4, KIA and KIB cells was
signi®catively higher than in parental K562 cells and
neo transfectants (Figure 3c). No major di€erences in
the extent of erythroid di€erentiation were found
between the D106-143 and In373 transfectants. The
percentage of benzidine-positive cells in the transfectants was similar to that of parental K562 cells treated
with ara-C. This drug induces erythroid di€erentiation
of K562 accompanied by irreversible growth arrest and
more pronounced erythroblastoid morphology (Rowley
et al., 1981). Ara-C induces an 8 ± 10-fold increase in
the fraction of hemoglobinized cells in K562 and Kneo
clones, but only 2 ± 3-fold in the D106-143 and In373
transfectants (Figure 3c).
Generation of cell lines transfected with inducible max
gene
Increased levels of Max would result in the higher
relative levels of Max/Max inactive homodimers that
Max and Myc in differentiation and apoptosis of K562 cells
M CanÄelles et al
1319
c
a
K562
C
A
KD2
C
A
KD4
C
A
KIA
C
A
KIB
C
A
-globin
∋
rRNAs
b
Figure 3 K562 sublines expresssing c-myc mutants show enhanced erythroid di€erentiation. (a) e-globin mRNA expression of
transfected cells. RNAs from the indicated cell lines were prepared from control cells (lanes `C') and cells treated for 3 days with
1 mM ara-C (lanes `A'). (b) Quanti®cation of e-globin mRNA signals of the above Northern blot with respect to the 18s rRNA. The
radioactivity of the signals was determined as described in the text and represented with respect to the maximum value. (c) Fraction
of benzidine-positive cells in transfectant cell lines. Benzidine test was carried out in untreated growing cells and cells treated for 3
days with 1 mM ara-C. The maximum score (KD2 cells treated with ara-C, 58%) was set at 100% and the other data were
normalized to this value. Bars indicate standard deviations from three separate experiments
compete with c-Myc/Max for common DNA binding
sites. To investigate the e€ects of Max overexpression
in K562 we transfected an expression vector where max
cDNA is under the control of murine metallothionein
promoter and can be induced by the addition of zinc to
the media. The plasmid was electroporated into K562
and transfectant cells were selected with hygromycin B
as described in Materials and methods. Two cell lines,
Kmax12 and Kmax16 were selected which showed a
marked increase in max mRNA (Figure 4a) and
protein (Figure 4b) upon addition of 75 mM ZnSO4.
The increase in max expression with zinc was dosedependent. Both cell lines express higher amounts of
max than parental cells in the absence of zinc addition,
due to the transcriptional leakiness of the metallothionein promoter.
di€erences were observed in the growth rate of
transfected cells upon addition of 50 mM ZnSO4 (not
shown). The addition of ara-C to Kmax12 and
Kmax16 resulted in a further increase in benzidinepositive cells (about 2-fold). This increase, however,
was much smaller than that observed for parental
K562 cells (8 ± 10-fold) (Figure 5). In contrast, we did
not detect any increase in the expression of markers of
myelomonocytic di€erentiation (reduction of nitroblue
tetrazolium, adherence, expression of GpIIb-IIIa) upon
induction of Max expression by zinc. Also, the
induction of Max expression did not alter the growth
rate of Kmax12 and Kmax16 (not shown). Addition of
zinc did not induce any erythroid di€erentiation in
KMMT cells, transfected with the empty vector
(pHEBoMT) (Figure 5).
Max overexpression enhances erythroid di€erentiation of
K562
Expression of c-Myc mutants impairs apoptosis of K562
To monitor changes in the extent of erythroid
di€erentiation, we determined the fraction of benzidine positive cells in Kmax12 and Kmax16 cells in the
presence and absence of zinc. When the expression of
Max was induced by 50 mM ZnSO4, a signi®cant
increase in the fraction of hemoglobinized cells was
observed for both cell lines, being higher for Kmax12
(Figure 5). The fraction of benzidine-positive cells after
zinc treatment was close to that observed for parental
cells treated with ara-C. An increased number of cells
with morphology compatible with that of basophilic
erythroblasts was also observed (not shown). No
As c-Myc mediates apoptosis in di€erent cell types
deprived of growth factors, we ®rst investigated
whether this e€ect is reproduced in K562 cells. Cells
were shifted to medium containing 2% or no serum for
24 or 48 h and apoptosis was assessed by DNA
fragmentation and scoring of apoptotic cells after
Giemsa staining. As shown in Figure 6a, K562 cells
showed some DNA degradation after 2 days of culture
in the absence of serum. The fraction of apoptotic cells
at this point was only 6% as determined by
morphological analysis of Giemsa-stained preparations. However, cells grew in the presence of 2% of
serum without any sign of apoptosis. We analysed
Max and Myc in differentiation and apoptosis of K562 cells
M CanÄelles et al
1320
a
Kmax12
0
25
Kmax16
50 100 200 0
25
50 100 200
µM Zn
max
rRNAs
b
Kmax12
0
2
6
12
Kmax16
24
0
2
6
12
24 hours
max
rRNAs
c
Kmax12
M
Kmax16
K562 0 12 24 48 0 12 24 48 hours
▲
27,5 —
Figure 5 Induction of max expression results in erythroid
di€erentiation. K562, KMMT, Kmax12 and Kmax16 cells were
exposed to either 50 mM ZnSO4, 1 mM ara-C or to 50 mM ZnSO4
for 4 h prior ara-C induction and then to 1 mM ara-C for three
days. Untreated cells were used as control. The maximum score
(Kmax12 cells treated with Zn+ara-C, 65%) was set at 100%
and the other data were normalized to this value. Bars indicate
standard deviations from three separate experiments
p21/22 Max
16,5 —
Figure 4 Induction of max expression in transfected K562 cell
lines. Northern analysis of max expression in Kmax12 and
Kmax16 sublines exposed to the indicated concentrations of
ZnSO4 for 4 h (a) or to 75 mM ZnSO4 for the indicated times (b).
Lower panels show the ®lters stained with ethidium bromide
showing the rRNAs. (c) Immunoblot of Max in K562, Kmax12
and Kmax16 cells exposed to 75 mM ZnSO4 for the indicated
times. Molecular mass markers (kDa) are indicated at the left
internucleosomal DNA fragmentation and the fraction
of apoptotic cells in KmycJ cells deprived of fetal calf
serum in the presence and absence of zinc. We found
that the overexpression of c-myc induced by 75 mM
ZnSO4 only resulted in a modest increase of apoptosis
as assessed by internucleosomal DNA fragmentation
(Figure 6a). The fraction of apoptotic cells after 2 days
of serum deprivation was about 4% and rose to 8% in
the presence of zinc (mean values from three
independent experiments). The same experiment was
performed with cells expressing c-Myc inhibitory
mutants, and we observed no signi®cant changes in
the extent of DNA fragmentation in KD2, KD4, KIA
and KIB with respect to parental cells after 3 days of
incubation in low-serum medium (Figure 6b). Therefore, the extent apoptosis induced by serum deprivation in K562 is low and shows little change by the
expression of c-Myc or inhibitory c-Myc mutant
proteins.
In view of the low level of apoptosis induced by
serum deprivation in K562 cells, we set out to study a
possible role of c-Myc in drug-induced apoptosis. K562
are remarkably resistant to apoptosis mediated by
many drugs, but okadaic acid (OA), a serine/threonine
a
K562
0%
KmycJ
2%
– – + + – – + +
M1 2 1 2 1 2 1 2
b
0%
2%
FCS
– – + + – – + + Zn
1 2 1 2 1 2 1 2 days
0% FCS
2% FCS
K562 KD2 KD4 KIA KIB
K562 KD2 KD4 KIA KIB
M 2 3 2 3 2 3 2 3 2 3
2 3 2 3 2 3 2 3 2 3 days
Figure 6 (a) Apoptosis of K562 and KmycJ cells incubated in
the absence of serum. K562 and KmycJ cells were grown for one
or two days in the absence or presence of 2% fetal calf serum
(FCS) and in the absence or presence of 75 mM ZnSO4 as
indicated at the top of the ®gure. Apoptosis was determined by
internucleosomal DNA fragmentation as described in the text. (b)
Apoptosis of cells expressing dominant negative c-myc mutants.
The indicated cell lines were treated for two or three days in the
absence or presence of 2% serum as indicated. Apoptosis was
determined by detection of internucleosomal DNA fragmentation
as above. Lane M refers to DNA molecular weight markers
(lambda DNA digested with EcoRI and HindIII)
Max and Myc in differentiation and apoptosis of K562 cells
M CanÄelles et al
1321
a
a
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K562 KD2 KIA KIB
Zn
OA
days
K562
KD4
M C OA C OA C OA C OA M C OA C OA
c-myc
9 10 11 12
1 2 3 4 5 6 7 8
rRNAs
b
120
–
+
3
+
+
1
+
+
2
+
+
3
+
–
1
+
–
2
+
–
3
Zn
OA
days
Figure 7 Apoptosis induced by OA during enforced expression
of c-myc. KmycJ cells were treated with 15 nM OA or 75 mM
ZnSO4 as indicated on the top of each lane. (a) Northern blot
showing c-myc expression in KmycJ cells. Filter was hybridized to
c-myc probe as indicated. A picture of the ®lter after transfer
shows the rRNAs stained with ethidium bromide. The size of the
exogenous c-myc mRNA is smaller than the endogenous one due
to the lack of the ®rst untranslated exon in the transfected
construct (Delgado et al., 1995). (b) Apoptosis detected by DNA
fragmentation assay. KmycJ cells were treated with 15 nM OA
and/or 75 mM ZnSO4 as indicated on the top of the picture and
the DNA was processed as indicated in Materials and methods.
Lane M shows DNA molecular weights markers (lambda DNA
digested with HindIII)
protein phosphatases inhibitor, can induce apoptosis in
K562 at nanomolar concentratioans (Zheng et al.,
1994; Lerga et al., 1995). We ®rst investigated whether
c-myc overexpression modi®ed the apoptosis induced
by OA in KmycJ cells. The results (Figure 7a)
con®rmed that the induction of c-myc mRNA
expression by 75 mM ZnSO4 was not impaired by
15 nM OA. The Northern blot of Figure 7a also shows
that the expression of endogenous c-myc mRNA was
slowly down-regulated following the OA treatment, as
previously described (Lerga et al., 1995). The extent of
apoptosis was determined by oligonucleosomal DNA
fragmentation and the results (Figure 7b) showed that
the ectopic expression of c-myc did not signi®cantly
modify the apoptosis provoked by OA. The same result
was found by scoring the fraction of morphologically
apoptotic cells, which was about 70% after 48 h of OA
treatment in the absence and presence of zinc (not
shown).
However, c-Myc could be required for OA to trigger
the apoptosis response in the cells. To investigate this
possibility we analysed the e€ect of c-Myc mutants on
OA-mediated apoptosis of K562 cells. KD2, KD4,
KIA and KIB cells were treated with 15 nM OA for
48 h and the level of apoptosis was determined by
genomic DNA fragmentation. A reduction was
observed in the apoptosis in the transfectants, as
compared to K562 cells (Figure 8a). This partial
inhibition of apoptosis was con®rmed by the fraction
of apoptotic cells after Giemsa staining (Figure 8b) and
the level of cytoplasmic nucleosomes, determined by
80
60
40
20
0
K562
Kneo2
Kneo3
Kneo6
Kneo8
KD2
KD4
KIA
KIB
–
+
2
K562
Kneo2
Kneo3
Kneo6
Kneo8
KD2
KD4
KIA
KIB
–
+
1
OA
UNTREATED
c
120
NUCLEOSOMS IN CYTOPLASM (%)
M
–
–
0
APOPTOTIC CELLS (%)
100
b
100
80
60
40
20
0
K562
KD2
KD4
KIA
KIB
Figure 8 Apoptosis induced by OA is reduced in cell sublines
expressing c-myc mutants. (a) Internucleosomal DNA fragmentation assay from control cells (lanes `C') and cells treated with
15 nM OA (lanes `OA'). Cells were treated for 48 h (lanes 1 to 8)
or for 36 h (lanes 9 to 12). Lanes M show DNA molecular
weights markers (lambda DNA digested with HindIII). (b)
Fraction of apoptotic cells, determined by morphological
analysis after Giemsa staining. Cells were treated for 48 h with
15 nM OA and the fraction of apoptotic cells was determined. The
fraction of apoptotic cells in the absence of OA was below 3% for
all cell lines. The maximum score (Kneo6 cells treated with OA,
80% apoptotic cells) was set at 100% and the other data were
normalized to this value. Bars indicate standard deviations from
three (Kneo, KD2, KD4) or four (K562, KIA and KIB) separate
experiments. (c) Apoptosis quanti®cation by the level of
cytoplasmic nucleosomes. Cells were treated for 48 h with
15 nM OA, lysed and the amounts of DNA-histone complexes
were determined as described in Materials and methods. The cell
lines tested are indicated
Max and Myc in differentiation and apoptosis of K562 cells
M CanÄelles et al
1322
Kmax16
Kmax12
KMMT
+
+
K562
Kmax12
Kmax16
+ + + + – – – – + + +
– – – – + + + + + + +
KMMT
K562
KMMT
Kmax16
Kmax12
– – – –
– – – –
K562
K562
KMMT
Kmax16
Kmax12
a
Zn
OA
b
Figure 9 Apoptosis induced by OA is reduced in max expressing
cell lines. (a) Internucleosomal DNA fragmentation assay for
K562, KMMT, Kmax 12 and Kmax16 cells. Cells were treated
for 48 h with 50 mM ZnSO4 (Zn) and 15 nM OA as indicated. (b)
Fraction of apoptotic cells, determined by morphological analysis
after Giemsa staining. Cells were treated for 48 h with 50 mM
ZnSO4 (Zn) in the presence or absence of 15 nM OA as indicated.
The fraction of apoptotic cells was determined after Giemsa
staining as indicated in Materials and methods. The maximum
score (KMMT treated with zinc and OA, 84% apoptotic cells)
was set at 100% and the other data were normalized to this value.
Bars indicate standard deviations from six separate experiments
ELISA (Figure 8c). The inhibition of apoptosis shown
by transfectant cells is only partial, as can be expected
from the low expression of mutant proteins (Figure 2c).
The average apoptotic cells for K562, Kneo2, Kneo3,
Kneo6 and Kneo8 (control cell lines) is 74.3+9.4%
(+s.d.), while the average for KD2, KD4, KIA and
KIB is 41.0+14.3%. Statistical analysis of the data
con®rms that the di€erence between control and
mutant c-myc transfected cells is signi®cative at a
97.3% con®dence level. We also assessed by Northern
analysis that the expression of D106-143 and In373 cmyc alleles was not modi®ed by the treatment with
15 nM OA (not shown).
Max overexpression impairs apoptosis of K562
We next studied whether Max overexpression modi®ed
the extent of apoptosis induced by OA in K562 cells.
Kmax12 and Kmax16 were treated with 15 nM OA in
the presence and absence of 50 mM ZnSO4 for 48 h and
the fragmentation of DNA was analysed. A representative experiment is shown in Figure 9a. The DNA
fragmentation induced by OA was reduced upon zinc
treatment in the Kmax16 and to a larger extent in
Kmax12 cells, as compared to K562 (parental cells)
and KMMT (cells transfected with the empty vector).
This result was con®rmed by determining the fraction
of apoptotic cells after OA treatment in the presence of
zinc. As shown in Figure 9b, a decrease in the
percentage of apoptotic cells was observed in
Kmax12 and Kmax16 cells treated with 50 mM
ZnSO4, with respect to K562 or KMMT cells. Again,
apoptosis supression was small for Kmax16 and very
signi®cant for Kmax12 cells, thus reproducing the
result observed in the internucleosomal DNA fragmentation assay. This result was parallel to that found for
erythroid di€erentiation, where Kmax12 was also the
most di€erentiated cell line. The fraction of apoptotic
cells was already reduced in transfected cells even in
the absence of zinc (Figure 9b), a result consistent with
the high basal expression of Max in these cells (Figure
4). However, some further reduction in apoptosis was
observed in the presence of zinc in max-transfected
cells. This reduction was small, but taking together the
results from the DNA fragmentation assay (Figure 9a)
and the fraction of apoptotic cells (Figure 9b), we
conclude that OA-mediated apoptosis decreased for
Kmax12 and Kmax16 cells in the presence of zinc. In
contrast, the extent of apoptosis was similar for K562
cells and KMMT cells in the absence or presence of
zinc, indicating that the decrease in apoptosis in
Kmax12 and Kmax16 was a consequence of the
induction of Max expression.
Discussion
The human myeloid leukemia K562 cell line provides a
unique model system where the biological functions of
c-Myc can be studied during di€erentiation towards
two separate lineages (erythroid and myelomonocytic)
and during drug-induced apoptosis. The e€ect of cMyc dominant negative mutants has been studied in
di€erentiation in murine preadipocytic and erythroleukemia cells (Freytag et al., 1990; Ohmori et al., 1993)
and apoptosis of murine ®broblasts (Evan et al., 1992).
However, to our knowledge the e€ects of c-Myc
mutants and Max have not been studied in differentiation and apoptosis in the same cell line. We have
carried out such study on human K562 myeloid cells.
We have found that the expression of either two
di€erent c-Myc mutants or ectopic Max overexpression
have an inhibitory e€ect on drug-mediated apoptosis of
K562 cells, and enhance erythroid di€erentiation, while
myelomonocytic di€erentiation was not modi®ed.
Among the c-Myc mutant proteins tested, D106-143
and In373 have been shown to be completely inactive
for all the biological activities of c-Myc as transformation of Rat-1a ®broblasts (Stone et al., 1987), apoptosis
of Rat-1 cells (Evan et al., 1992), DNA synthesis of
NIH3T3 cells (Goruppi et al., 1994), and REC
cotransformation with Ras (Stone et al., 1987). Both
D106-143 and In373 act as dominant negative c-Myc
mutants in transformation of Rat-1 cells mediated by
Max and Myc in differentiation and apoptosis of K562 cells
M CanÄelles et al
Bcr/Abl or v-Abl (Sawyers et al., 1992) and D106-143
in the Myc-Ras cotransformation of REC (Dang et al.,
1989). Conversely to wild-type c-myc, these mutants
are unable to inhibit di€erentiation of murine
erythroleukemia cells (Bar-Ner et al., 1992; Ohmori et
al., 1993) and preadipocytic cells (Freytag et al., 1990).
The deletion of the D106-143 protein includes the Myc
box II (amino acids 122 ± 143), a region conserved
amongst Myc family members. This box is required for
cell transformation and transcriptional repression
dependent on initiator (Inr) element of promoters,
but seems to be dispensable for transactivation (Kato
et al., 1990; Amin et al., 1993; Li et al., 1994; Brough
et al., 1995; Lee et al., 1996). So it is likely that D106143 inhibits Myc function by interfering with not only
transactivation but also with the transcriptional
repression activity of c-Myc. In contrast, the potent
dominant negative mutant In373 carries an insertion in
the ®rst helix of the HLH domain in the C-terminal
part of c-Myc. We have shown that In373 is able to
dimerize with Max in vivo, interrupts DNA binding by
Myc/Max heterodimers and inhibits Myc cotransforming ability in rat embryo cells (Figure 1) thus acting as
dominant negative c-Myc protein in this assay. These
data con®rm a recent report showing that In373
inhibits Myc/Max binding to DNA in reticulocytes
lysates (Katzav et al., 1995). Therefore, our results
strongly indicate that the biochemical basis of In373
function is disruption of Myc/Max DNA binding. The
common biochemical activity of D106-143 and In373 is
to bind Max and hence to titrate out c-Myc to form
non-functional Myc/Max dimers.
E€ects on growth and di€erentiation
We generated K562 stably transfected with the D106143 and In373 mutant c-myc genes. The expression of
both mutants impaired K562 growth, as expected from
the requirement of c-Myc in cell proliferation (Heikkila
et al., 1987; Prochownik et al., 1988; Eilers et al., 1991;
Evan et al., 1992; Nguyen et al., 1995). However, we
wanted to isolate stable transfectants constitutively
expressing the c-Myc inhibitory mutants so as to study
their e€ects on di€erentiation and apoptosis of K562
cells. Cell lines transfected with D106-143 contained
multiple copies of the gene and expressed high levels of
c-myc mRNA, but the immunoblots detected lower
levels of D106-143 with respect to endogenous wildtype c-Myc. The levels of In373 in the transfectants
could not be determined but we believe they are also
low as no signi®cant increase in the total c-Myc protein
band was detected in Western blots (not shown). This
result could be expected from the growth inhibitory
e€ect of the D106-143 and In373 c-Myc mutants.
Therefore, only those clones expressing low amounts of
mutant c-Myc proteins will be selected after long-term
culture. Consistently, the stable transfectants obtained
showed a slightly reduced growth rate with respect to
parental cells. It has been described the presence of
transcripts of inhibitory mutant c-myc genes in stable
transfectants (Stone et al., 1987; Mukherjee et al.,
1992) and the expression of mutant c-Myc proteins
after transient transfection (Stone et al., 1987).
However, it is reportedly dicult to document the
presence of these mutant c-Myc proteins in stably
transfected cells (Stone et al., 1987; Freytag et al.,
1990; Sawyers et al., 1992), despite phenotypic changes
described for the transfectants. To our knowledge, the
K562 transfectants reported here and the Friend
erythroleukemia cells (Bar-Ner et al., 1992) constitute
the only examples where the constitutive expression of
a dominant negative c-Myc mutant is documented in
stably transfected cells. This is probably due to the
high basal expression of normal c-myc in these cell
lines, which allows the expression of limited amounts
of inhibitory c-Myc mutants.
We took advantage of the K562 model to
investigate c-Myc involvement in di€erentiation and
apoptosis. We have previously shown that K562 cells
show a high level of basal c-myc expression and this
expression is down-regulated when the cells are
induced to di€erentiate towards erythroid and
myelomonocytic lineages (Gomez-Casares et al.,
1993). Therefore we asked whether the expression of
c-myc inhibitory mutants and the overexpression of
max interferes with di€erentiation of K562 cells. We
show in this paper that the expression of D106-143
mutant, of In373 mutant and the overexpression of
Max conferred signs of erythroid di€erentiation in
human myeloid cells. The three proteins antagonize
the e€ects of c-Myc in proliferation assays. Now we
extend this fact to a di€erentiation model system.
Both c-Myc mutants sequester Max so as to form
heterodimers that are expected to be biologically
inactive. Therefore, it is conceivable that Max levels
are limiting in K562, and the dimerization of Max
with D106-143 or In373 results in a decrease of wildtype Myc/Max heterodimers. The induction of Max in
cells transfected with the inducible gene would result
in increased levels of Max/Max dimers, competing
with Myc/Max heterodimers for their molecular
targets. In agreement with the results presented here,
it has been previously observed that ectopic c-myc
overexpression partially inhibits erythroid differentiation of K562 cells (Baker et al., 1994; Delgado et al.,
1995). In murine erythroleukemia cells the ectopic
expression of c-Myc inhibits di€erentiation, while
overexpression of Max did not increase the differentiation (Dunn et al., 1994). This discrepancy with
our results could be explained again if basal Max
levels relative to c-Myc were lower in K562 cells than
in murine erythroleukemia cells.
We cannot rule out the possibility that the biological
responses described in the transfectant K562 cells are
due to mechanisms independent of c-Myc. However,
the common molecular consequence of the expression
of D106-143, In373 and Max would be to decrease the
amount of active c-Myc/Max dimers bound to their
DNA targets. The mechanism by which lowering the
levels of c-Myc/Max would enhance erythroid differentiation of K562 is unknown. One possibility is that
the primary e€ect is to impair the growth of K562 and
the di€erentiation would come as a consequence of
this. However, the fact that growth arrest by low
serum, high cell density (our unpublished observations)
or treatment by interferons (GoÂmez-Casares et al.,
1993) do not result in di€erentiation argues against the
idea that growth arrest per se is sucient for K562
erythroid di€erentiation. Also, Kmax12 and Kmax16
cells grow at similar rates in the absence and presence
of zinc, while erythroid di€erentiation is enhanced in
the latter case.
1323
Max and Myc in differentiation and apoptosis of K562 cells
M CanÄelles et al
1324
K562 cells are bipotential, as can di€erentiate into
erythroid or myelomonocytic lineages. Thus this
system could allow the detection of a di€erential
role of c-Myc depending on the di€erentiation
pathway. It is noteworthy that we did not detect
any signi®cant change in the expression of myelomonocytic markers in cells transfected with c-myc
mutants or with inducible max after zinc addition.
Also, the extent of myelomonocytic di€erentiation
induced by TPA was similar for the transfectants and
the parental cells. Consistently with this result, we
previously found that ectopic c-myc overexpression
does not modify TPA-mediated myelomonocytic
di€erentiation of K562 (Delgado et al., 1995). To
our knowledge this is the ®rst example of a
di€erential involvement of c-Myc in two differentiation pathways of the same cell type.
E€ect on apoptosis
Myc-mediated apoptosis has been explained by
proposing that apoptosis is a physiological function
of c-Myc but normally inhibited by growth factors
(Harrington et al., 1994; Packham and Cleveland,
1995). The apoptotic function of c-Myc requires
dimerization with Max (Amati et al., 1993b; Bissonnette et al., 1994). We have found that K562 cells are
relatively resistant to apoptosis by serum deprivation
and the overexpression of c-Myc only slightly increased
this apoptosis (Figure 6). This is in sharp contrast with
the dramatic apoptotic response to c-Myc overexpression of ®broblasts and myeloid cells deprived from
growth factors (Askew et al., 1991; Evan et al., 1992).
Consistently, expression of inhibitory c-Myc mutants
did not decrease the apoptosis mediated by serum
deprivation. We conclude that the cell death induced
by growth factors deprivation in K562 is to a large
extent Myc-independent. K562 are also remarkably
resistant to apoptosis induced by drugs as compared to
other human myeloid cell lines (Martin et al., 1990;
Ritke et al., 1994; McGahon et al., 1994; Benito et al.,
1995). However, apoptosis is readily induced in K562
by OA (Zheng et al., 1994; Lerga et al., 1995). OA
inhibits protein serine/threonine phosphatases type 1
and 2A, being more active against type 2A (reviewed in
Cohen et al., 1990; Wera and Hemmings, 1995). We
have previously shown that OA is capable of inducing
apoptosis in K562 at concentrations (15 nM, used in
the present work) that inhibit phosphatase type 2A but
not type 1 (Lerga et al., 1995). The apoptosis mediated
by OA in K562 is not further augmented by c-Myc
overexpression, which is in agreement with the high
basal level of c-Myc expression in K562 cells. However,
by all criteria analysed, the extent of OA-mediated
apoptosis was reduced in cells expressing the mutant cMyc proteins D106-143 and In373 as well as in cells
where Max is overexpressed. The result suggests that a
reduction in active c-Myc/Max complexes is responsible for the decreased apoptotic response to OA. We
conclude that c-Myc is involved in this apoptotic
process triggered by protein phosphatase inhibition.
We have found that OA-mediated apoptosis is
accompanied by slow down-regulation of c-myc and
max, but their mRNA and protein are still detectable
after 24 h of treatment with OA, when cells are
committed to apoptosis (Lerga et al., 1995; and this
work, Figure 7). Therefore, c-Myc could be required at
the earlier stages of the process (the `condemnation'
phase) (Earnshaw, 1995) and the expression of mutant
c-myc genes will impair the apoptosis mediated by OA.
The mechanism by which OA induces apoptosis is
unknown, although this e€ect is common to di€erent
cell types (Boe et al., 1991; Ishida et al., 1992; Kiguchi
et al., 1994). It has been reported recently that cdc25A
is a target gene for c-Myc and that cdc25A can induce
apoptosis in ®broblasts (Galaktionov et al., 1996). On
the other hand, OA can activate cdc25A through
protein phosphatase 2A inhibition (reviewed in Wera
and Hemmings, 1995). These results suggest that
cdc25A could be one of the mediators of the apoptotic
e€ect of OA and that Max and inhibitory c-Myc
mutants may counteract the up-regulation of cdc25A
mediated by c-Myc. Further work is required to test
this hypothesis. Taken together, our results support a
model where a threshold level of functional c-Myc/
Max dimers is required to maintain the cells in an
undi€erentiated state and to trigger an apoptotic
response to drug treatment.
Materials and methods
Bacterially synthesized proteins
Truncated c-Myc (tMyc; amino acids 342 ± 439) and Max
proteins were produced as previously described (Kato et
al., 1992). The truncated mutant Myc protein, tIn373, was
produced from the expression vector pDS-In373 that
encodes amino acids 343 ± 439 of c-Myc with an insert of
four serines at position 373. pDS-In373 was generated by
subcloning a blunt HindIII fragment from a polymerase
chain reaction ampli®ed fragment using Sp65MycA2-In373
(Stone et al., 1987) as the template. The primers were T3
and 5'-ACCAGCCCAGG TCCTCGG-3' (human c-myc
sequence starting at codon 343). The polylinker sequence
of pDS-Myc(342 ± 439) contributes to amino acids that
exceed the tIn373 polypeptide encoded by pDS-In373 by
two amino acids. All proteins are tagged with a
hexahistidine tail allowing for puri®cation over nickel
agarose as described previously (Kato et al., 1992).
Electrophoretic mobility shift assay (EMSA)
EMSAs were performed as previously described with a
CACGTG containing radiolabeled 260 bp probe pDW14
(Wechsler and Dang, 1992). This probe, in contrast to
shorter oligonucleotides, allows resolution of the mobilities
of the di€erent tMyc and Mac dimers.
Mammalian two-hybrid assay
The two-hybrid assay was performed as described (Dang
et al., 1991). The constructs pGal-Myc or GM(262 ± 439),
pNLVP, pVPMax(8 ± 112) and the reporter G5-E1b-CAT
were described previously (Kato et al., 1990). pGal-In373
was constructed from the mutant In373 (Stone et al.,
1987) by ligating a Myc-In373 ClaI ± NsiI fragment into
the GAL4 vector pGALO as described for GM(262 ± 439)
(Kato et al., 1990). Chinese hamster ovary (CHO) cells
were transfected with 2 or 4 mg of activator DNA and
2 mg of reporter using DEAE dextran as described (Kato
et al., 1990). Assays for chloramphenicol acetyltransferase
(CAT) activities were described previously (Kato et al.,
1990).
Max and Myc in differentiation and apoptosis of K562 cells
M CanÄelles et al
Mammalian expression c-myc and max plasmids
The plasmids pMLV-D106-143, pMLV-In373 and pMLVIn6 carrying mutant c-myc genes have been previously
described (Stone et al., 1987). D106-143 carries a deletion
between the 106 and 143 amino acids of c-Myc and In373
gene carries an insertion of four serines between amino
acids 373 and 374 of human c-Myc. In6 carries an insertion
between amino acid 6 and 7, and is used as an inactive
insertion mutant (Stone et al., 1987). pM21 encodes the
normal human c-myc gene. All these genes are under the
control of the Moloney murine leukemia virus LTR (Stone
et al., 1987). The vector pHEBoMT (kindly provided by F
Grignani) carries the mouse metallothionein I promoter
and the hygromycin resistance gene (Grignani et al., 1990).
Plasmid pHEBoMT-max was constructed by ligation of the
1.9 kb EcoRI insert of plasmid pBSmax (kindly provided
by R Dalla-Favera) into the BamHI site of the pHEBoMT
plasmid after blunt-ending the EcoRI and BamHI sites
with the Klenow fragment. pHEBoMT-max encodes the
human p21 Max under the control of the metallothionein
promoter.
Rat embryo cell cotransformation assay
The myc ± ras cotransformation assays were performed as
described previously (Dang et al., 1989). Transformed foci
were determined from quadruplicate experiments at 3
weeks after lipofection of rat embryo ®broblasts.
K562 cell lines and transfection
K562 cells were obtained from ATCC. KmycJ are K562
cells stably transfected with a zinc-inducible c-myc gene,
under the control of the mouse metallothionein I promoter
(Delgado et al., 1995). KMMT are K562 cells stably
transfected with the vector pHEBoMT (Delgado et al.,
1995). Cells were grown in RPMI-1640 medium (Whittaker) supplemented with 8% fetal calf serum (Biochrom)
and gentamicin (80 mg/ml). KmycJ and KMMT were
grown in the presence of 0.4 mg/ml of G418. 50 mg of
pMLV-In373 and pMLV-D106-143 plasmids (Stone et al.,
1987) were transfected along with 5 mg of pSV2neo into
K562 cells by electroporation (400 v, 500 mF) using a BioRad Gene Pulser apparatus. After electroporation the cells
were incubated for 48 h and 0.6 mg/ml of G418 was added.
40 mg of pHEBoMT-max plasmid were also transfected by
electroporation and selected in 0.2 mg/ml of hygromycin B.
Clones were obtained from the G418-resistant and
hygromycin-resistant pools by limiting dilution in microtiter wells and were expanded and analysed. To be used as
controls, K562 cells were also transfected with a plasmid
expressing the G418 resistance gene (pMAMneo, Clontech)
and selected as indicated above.
Assessment of cell growth rate and di€erentiation
Cell growth and viability were assayed by hemocytometer
and the trypan blue exclusion test. Erythroid differentiation was induced by adding to growing cells 1 mM ara-C.
The fraction of hemoglobin-producing cells was scored by
the benzidine assay essentially as described (Rowley et al.,
1981). Myelomonocytic di€erentiation was induced by
treating the cells with 10 nM TPA. The di€erentiation
was determined by cell clustering activity, the ability to
reduce nitroblue tetrazolium and the expression of
glycoprotein IIb-IIIa, determined by ¯ow cytometry as
described (Delgado et al., 1992).
DNA and RNA analysis
Cellular DNA was isolated by lysing cells in a solution
containing 10 mM Tris (pH 8), 150 mM NaCl, 3 mM EDTA
with addition of 0.5% SDS and 0.4 mg/ml proteinase K.
After an overnight incubation at 378C, DNA was extracted
by precipitation with 0.4 M NaCl and isopropanol and
dissolved in TE. 15 mg of DNA were treated with EcoRI,
separated in 0.7% agarose gel and transferred to
nitrocellulose membranes (Millipore) essentially as described (Sambrook et al., 1989). Total RNA was isolated
from cells by the acid guanidine thiocyanate method
(Chomczynski and Sacchi, 1988). RNA samples (25 mg
per lane) were electrophoresed on 1% agarose-formaldehyde gels and transferred to nitrocellulose membranes
(Millipore) as described (Sambrook et al., 1989) except that
1 mg of ethidium bromide was added to each RNA sample
prior to gel loading. A picture of the ®lter under u.v. light
was obtained after transfer to assess the amount and
integrity of the rRNAs. The Southern and Northern blots
were hybridized at 428C in 40% formamide, 5% dextran
sulphate, 0.8 M NaCl, 50 mM sodium phosphate (pH 7),
0.2% SDS, 150 mg of denatured salmon sperm DNA per ml
and DNA probe labeled with [32P]dCTP by random primed
labeling (Pharmacia kit). The ®lters were washed to a ®nal
stringency of 0.56SSC and 0.1% SDS at 658C and
autoradiographed at 7708C. The signals were quanti®ed
with a Bio-Rad Molecular Imager apparatus. Probes for
human c-myc, max and e-globin genes were as described
(Delgado et al., 1995).
Immunoblots
Cell pellets were lysed in a solution containing
100 mM Tris (pH 6,8), 8% b-mercaptoethanol, 4% SDS
and 20% glycerine. Protein content was measured using
Bio-Rad Protein Assay. 40 mg of protein per lane were
separated in 10% acrylamide gels and transferred to PVDF
membranes (Immobilon, Millipore), using a semi-dry
electroblotter (Millipore) and a bu€er containing 10 mM
Trizma, 96 mM glycine and 10% methanol. c-Myc and
Max proteins were detected by the anti-Myc monoclonal
antibody 9E10 (Oncogene Science) or the anti-Max
polyclonal antibody (UBI). Membranes were then developed using chemiluminescent detection (Tropix).
Apoptosis determinations
For analysis of internucleosomal DNA fragmentation
(Duke and Cohen, 1986), cells were lysed in a solution
containing 10 mM Tris, 1 mM EDTA and 0.2% Triton X100. Cytoplasmic fraction of the lysates was obtained by
centrifugation and further adjusted to 150 mM NaCl,
40 mM EDTA, 1% SDS and treated with 200 mg/ml of
proteinase K. DNA fragments were precipitated with
ethanol and separated in 1.5% agarose gels containing
0.1 mg/ml ethidium bromide. Quanti®cation of apoptosis
by determination of cytoplasmic nucleosomes was carried
out by the Cell Death Detection ELISA assay (Boehringer
Mannheim). Cytospin preparations were stained by Giemsa
and the fraction of apoptotic cells was scored under the
microscope. Apoptotic cells were identi®ed by chromatin
condensation and marginalization, nuclear fragmentation
and cytoplasmic shrinkage. At least 200 cells were analysed
for each point by observers without knowledge of the
sample analysed.
Acknowledgements
We thank Pilar Frade for tissue culture and photographic
work, Robert Eisenman and Steven Collins for critical
reading of the manuscript and helpful advice, Franco
Grignani, Riccardo Dalla-Favera, Robert Eisenman and
Waturu Shoji for clones, Narciso Benitez for statistical
analysis and illustrations design and M Luisa GarcõÂ a for
technical assistance. This work was supported by grants
1325
Max and Myc in differentiation and apoptosis of K562 cells
M CanÄelles et al
1326
DGICYT PB92-506CO-2 and CICYT SAF96-0083 from
Spanish Government, a grant from FundacioÂn RamoÂn
Areces and NIH grant CA51497. MC was supported by a
fellowship from Gobierno Vasco, KMH by NIH training
grant T32GM07814 and AL by a FPI fellowship from
Ministerio de EducacioÂn y Ciencia, Spain.
References
Amati B and Land H. (1994). Curr. Op. Genet. Develop., 4,
102 ± 108.
Amati B, Brooks MW, Levy N, Littlewood TD, Evan GI and
Land H. (1993a). Cell, 72, 233 ± 245.
Amati B, Littlewood TD, Evan GI and Land H. (1993b).
EMBO J., 12, 5083 ± 5087.
Amin C, Wagner AJ and Hay N. (1993). Mol. Cell. Biol., 13,
383 ± 390.
Askew DS, Ashmun RA, Simmons BC and Cleveland JL.
(1991). Oncogene, 6, 1915 ± 1922.
Baker SJ, Pawlita M, Leutz A and Hoelzer D. (1994).
Leukemia, 8, 1309 ± 1317.
Bar-Ner M, Messing LT, Cultraro CM, Birrer MJ and Segal
S. (1992). Cell Growth Di€., 3, 183 ± 190.
Benito A, Grillot D, NunÄez G and FernaÂndez-Luna JL.
(1995). Am. J. Pathol., 146, 481 ± 490.
Bissonnette RP, McGahon A, Mahboubi A and Green DR.
(1994). J. Exp. Med., 180, 2413 ± 2418.
Blackwood EM and Eisenman RN. (1991). Science, 251,
1211 ± 1217.
Blackwood EM, Kretzner L and Eisenman RN. (1992). Curr.
Op. Genet. Develop., 2, 227 ± 235.
Boe R, Gjertsen BT, Vintermyr OK, Houge G, Lanotte M
and Doskeland SO. (1991). Exp. Cell Res., 195, 237 ± 246.
Brough DE, Hofmann TJ, Ellwood KB, Townley RA and
Cole MD. (1995). Mol. Cell. Biol., 15, 1536 ± 1544.
Charnay P and Maniatis T. (1983). Science, 220, 1281 ± 1283.
Chisholm O, Stapleton P and Symonds G. (1992). Oncogene,
7, 1827 ± 1836.
Chomczynski P and Sacchi N. (1988). Anal Biochem., 162,
156 ± 159.
Cohen P, Holmes CFB and Tsukitani Y. (1990). Trends
Biochem. Sci., 15, 98 ± 102.
Coppola JA and Cole MD. (1986). Nature, 320, 760 ± 763.
Dang CV, Barrett J, Villa-GarcõÂ a M, Resar LMS, Kato GJ
and Fearon ER. (1991). Mol. Cell. Biol., 11, 954 ± 962.
Dang CV, McGuire M, Buckmire M and Lee WMF. (1989).
Nature, 337, 664 ± 666.
Delgado MD, Quincoces AF, GoÂmez-Casares MT, MartõÂ nez
CA, Cuadrado MA, Richard C and LeoÂn J. (1992). Cancer
Res., 52, 5948 ± 5979.
Delgado MD, Lerga A, CanÄelles M, GoÂmez-Casares MT and
LeoÂn J. (1995). Oncogene, 10, 1659 ± 1665.
Dmitrovski E, Kuelk WM, Hollis GF, Kirsch IR, Bender TP
and Segal S. (1986). Nature, 322, 748 ± 750.
Duke RC and Cohen JJ. (1986). Lymphokine Res., 5, 289 ±
299.
Dunn BK, Cogliati T, Cultraro CM, Bar-Ner M and Segal S.
(1994). Cell Growth Di€er., 5, 847 ± 854.
Earnshaw WC. (1995). Curr. Op. Cell Biol., 7, 337 ± 343.
Eilers M, Schirm S and Bishop JM. (1991). EMBO J., 10,
133 ± 141.
Evan GI, Wyllie AH, Gilbert CS, Littlewood TD, Land H,
Brooks M, Waters CM, Penn LZ and Hancock DC.
(1992). Cell, 69, 119 ± 128.
Evan GI and Littlewood TD. (1993). Curr. Op. Genet.
Develop., 3, 44 ± 49.
Freytag SO, Dang CV and Lee WMF. (1990). Cell Growth
Di€er., 1, 339 ± 343.
Galaktionov K, Chen X and Beach D. (1996). Nature, 382,
511 ± 517.
GoÂmez-Casares MT, Delgado MD, Lerga A, Crespo P,
Quincoces AF, Richard C and Leo n J. (1993). Leukemia,
7, 1824 ± 1833.
Goruppi S, Gustincich S, Brancolini C, Lee WMF and
Schneider C. (1994). Oncogene, 9, 1537 ± 1544.
Grignani F, Lombardi L, Inghirami G, Sternas L, Cechova
K and Dalla-Favera R. (1990). EMBO J., 9, 3913 ± 3922.
Gu W, Cechova K, Tassi V and Dalla-Favera R. (1993).
Proc. Natl. Acad. Sci. USA, 90, 2935 ± 2939.
Harrington EA, Fanidi A and Evan GI. (1994). Curr. Op.
Genet. Develop., 4, 120 ± 129.
Heikkila R, Schwab G, Wickstrom E, Loke SL, Pluznik DH,
Watt R and Neckers LM. (1987). Nature, 328, 445 ± 449.
Holt JT, Redner RL and Nienhuis AW. (1988). Mol. Cell.
Biol., 8, 963 ± 973.
Ishida Y, Furukawa Y, Decaprio JA, Saito M and Grin
JD. (1992). J. Cell. Physiol., 150, 484 ± 492.
Kamano H, Onnishi H, Tanaka T, Ikeda K, Okabe A and
Irino S. (1990). Leuk. Res., 14, 831 ± 839.
Kato GJ, Barret J, Villa-GarcõÂ a M and Dang CV. (1990).
Mol. Cell. Biol., 10, 5914 ± 5920.
Kato GJ, Lee WMF, Chen L and Dang CV. (1992). Genes
Develop., 6, 81 ± 92.
Kato GJ and Dang CV. (1992). FASEB J., 6, 3065 ± 3072.
Katzav S, Pakham G, Sutherland M, Aroca P, Santos E and
Cleveland JL. (1995). Oncogene, 11, 1079 ± 1088.
Kiguchi K, Glesne D, Chubb CH, Fujiki H and Huberman
E. (1994). Cell Growth Di€er., 5, 995 ± 1004.
Koskinen PJ, VaÈstrik I, MaÈkelaÈ TP, Eisenman RN and
Alitalo K. (1994). Cell Growth Di€er., 5, 313 ± 320.
Kretzner L, Blackwood EM and Eisenman RN. (1992).
Nature, 359, 426 ± 429.
Larsson LG, Ivhed I, Gidlund M, Pettersson U, VennstroÈm
B and Nilsson K. (1988). Proc. Natl. Acad. Sci. USA, 85,
2638 ± 2642.
Lee LL, Dolde C, Barrett J, Wu CS and Chi D. (1996). J.
Clin. Invest., 97, 1687 ± 1695.
Lerga A, Belandia B, Delgado MD, Cuadrado MA, Richard
C, Ortiz JM, MartõÂ n-Perez J and LeoÂn J. (1995). Biochem.
Biophys. Res. Commun., 215, 889 ± 895.
Li L, Nerlov C, Prendergast G, MacGregor D and Zi€ EB.
(1994). EMBO J., 13, 4070 ± 4079.
Lindeman GJ, Harris AW, Bath ML, Eisenman RN and
Adams JM. (1995). Oncogene, 10, 1013 ± 1017.
Lotem J and Sachs L. (1993). Cell Growth Di€er., 4, 41 ± 47.
MaÈkelaÈ TP, Koskinen PJ, VaÈstrik I and Alitalo K. (1992).
Science, 256, 373 ± 377.
McGahon A, Bissonnette RP, Schmitt M, Cotter KM, Green
DR and Cotter TG. (1994). Blood, 83, 1179 ± 1187.
Marcu KB, Bossone SA and Patel AJ. (1992). Annu. Rev.
Biochem., 61, 809 ± 860.
Martin S, Lennon S, Bonham A and Cotter T. (1990). J.
Immunol., 145, 1859 ± 1867.
Meichle A, Philipp A and Eilers M. (1992). Biochim. Biophys.
Acta., 1114, 129 ± 146.
Mukherjee B, Morgenbesser SD and DePinho R. (1992).
Genes Dev., 6, 1480 ± 1492.
Nguyen HQ, Selvakumaran M, Liebermann DA and
Ho€man B. (1995). Oncogene, 11, 2439 ± 2444.
Ohmori Y, Jun-ichi T, Takada S, Lee WMF and Obinata M.
(1993). Oncogene, 8, 379 ± 386.
Packham G and Cleveland JL. (1995). Biochim. Biophys.
Acta., 1242, 11 ± 28.
Prendergast GC, Lawe D and Zi€ EB. (1991). Cell, 65, 395 ±
407.
Prochownik EV, Kukowska J and Rodgers C. (1988). Mol.
Cell. Biol., 8, 3683 ± 3695.
Max and Myc in differentiation and apoptosis of K562 cells
M CanÄelles et al
Ritke MK, Bergoltz VV, Allan WP and Yalowich JC. (1994).
Biochem. Pharmacol., 48, 525 ± 533.
Rowley PT, Ohlsson-Wilhelm BM, Farley BA and LaBella S.
(1981). Exp. Hematol., 9, 32 ± 37.
Sambrook J, Fritsch EF and Maniatis T. (1989). Molecular
Cloning: A Laboratory Manual, 2nd edition. Cold Spring
Harbor Laboratory. Cold Spring Harbor, New York.
Sawyers CL, Callahan W and Witte ON. (1992). Cell, 70,
901 ± 910.
Selvakumaran M, Lin HK, Sjin RTT, Reed JC, Liebermann
DA and Ho€man B. (1994). Mol. Cell. Biol., 14, 2352 ±
2360.
Shen W-F, Detmer K, Mayhews CHE, Hack FM, Morgan
DA, Largman C and Lawrence HJ. (1992). EMBO J., 11,
983 ± 989.
Stone J, De Lange T, Ramsay G, Jakobovits E, Bishop JM,
Varmus H and Lee W. (1987). Mol. Cell. Biol., 7, 1697 ±
1709.
VaÈstrik I, MaÈkelaÈ TM, Koskinen PJ, Klefstrom J and Alitalo
K. (1994). Crit. Rev. Oncogenesis, 5, 59 ± 68.
Wechsler DS and Dang CV. (1992). Proc. Natl. Acad. Sci.
USA, 89, 7635 ± 7639.
Wera S and Hemmings BA. (1995). Biochem. J., 311, 17 ± 29.
Zheng B, Chambers TC, Raynor RL, Markham PN, Gebel
HM, Vogler WR and Kuo JF. (1994). J. Biol. Chem., 269,
12332 ± 12338.
1327