(Received for publication, July 13, 1995; and in revised form, September 15, 1995)
From the
The cellular proto-oncogene c-myc is involved in cell proliferation and transformation but is also implicated in the induction of programmed cell death (apoptosis). The c-Myc protein is a transcriptional activator with a carboxyl-terminal basic region/helix-loop-helix (HLH)/leucine zipper (LZ) domain. It forms heterodimers with the HLH/LZ protein Max and transactivates gene expression after binding DNA E-box elements. We have studied the phenotype of dominant-negative mutants of c-Myc and Max in microinjection experiments. Max mutants with a deleted or mutated basic region inhibited DNA synthesis in serum-stimulated 3T3-L1 mouse fibroblasts. In contrast, mutants of c-Myc expressing only the basic region/HLH/LZ or HLH/LZ domains rapidly induced apoptosis at low and high serum levels. Co-expression of the HLH/LZ domains of c-Myc and Max failed to do so. We suggest that the c-Myc HLH/LZ domain induces apoptosis by specific interaction with cellular factors different to Max.
Programmed cell death is an intrinsic death program operating to eliminate unwanted cells during normal development. It also has been suggested to kill cells after aquirement of growth factor-independent growth properties due to genetic alterations (Ellis et al., 1991; Evan and Littlewood, 1993). Common morphological features of programmed cell death are blebbing of the cytoplasmic membrane, chromatin condensation, and breaking of the dead cell into apoptotic bodies (Wyllie, 1980, 1987). This kind of cell death, often termed apoptosis, does not elicit an inflammatory response in the tissue and can therefore clearly be distinguished from cell necrosis in which cells die as the result of acute injury (Kerr et al., 1972).
The apoptotic program appears to be installed in all animal cells and can operate in the presence of inhibitors of RNA and protein synthesis (Ellis et al., 1991). In some cellular systems it is even induced by these inhibitors, indicating that short-lived proteins or RNAs may negatively control the apoptotic machinery. Jacobson et al.(1994) recently proposed a model in which the process leading to apoptosis is divided into three phases: (i) an activation phase in which control systems of apoptosis are activated or derepressed (this phase can be sensitive to inhibitors of RNA and protein synthesis), (ii) an effector phase in which the activated control system acts on multiple targets in the cell, and (iii) a degradation phase in which the dying cell is broken down. In the latter two phases, inhibitors of RNA and protein synthesis are not effective to block the apoptotic program.
The cellular proto-oncogene c-myc has been implicated in the control of proliferation and apoptosis. Expression of c-myc is tightly linked to mitogenic stimuli and is a prerequisite for cell growth (for reviews, see Lüscher and Eisenman(1990) and Marcu et al., 1992). Moreover, post-translational activation of a c-Myc estrogen receptor chimera in resting cells is sufficient to induce entry into the cell cycle (Eilers et al., 1989, 1991). Expression of an exogenous c-myc gene renders hematopoietic cells and fibroblasts unable to exit from the cell cycle upon withdrawal of growth factors or serum. Instead, these cells continue cycling and concomitantly undergo apoptosis (Askew et al., 1991; Evan et al., 1992; Hermeking and Eick, 1994). Expression of c-myc is also required for activation-induced apoptosis of T-cell hybridomas (Shi et al., 1992). These observations suggest a model in which proliferation and cell death are processes that are co-induced by c-Myc and which are subsequently modulated by cytokine action (Evan and Littlewood, 1993; Harrington et al., 1994).
The c-Myc protein has features of a transcription factor with
a transcriptional activation domain in the amino-terminal region (Kato et al., 1990). The carboxyl-terminal region contains a basic
region (BR), ()helix-loop-helix (HLH), and leucine zipper
(LZ) domain (Murre et al., 1989; Landschulz et al.,
1988) in a contiguous array essential for specific DNA binding (BR) and
dimerization (HLH/LZ) of c-Myc with the BR/HLH/LZ protein Max
(Blackwood and Eisenman, 1991; Blackwood et al., 1992; Kato et al., 1992). c-Myc
Max heterodimers and Max
Max
homodimers bind specifically to the E-box motif CACGTG (Blackwell et al., 1990). Homodimers of c-Myc are not found in
vivo. Since Max lacks a transcriptional activation domain,
c-Myc
Max heterodimers have been suggested to act as
transcriptional activator and Max
Max homodimers as repressors
(Kretzner et al., 1992; Amati et al., 1992). The
biological functions of c-Myc reported so far including cell
transformation (Stone et al., 1987, Amati et al.,
1993a), transcriptional activation (Kretzner et al., 1992;
Amati et al., 1993b), and induction of proliferation and
apoptosis in quiescent cells (Evan et al., 1992; Amati et
al., 1993b) require dimerization of c-Myc with Max and
sequence-specific binding of the heterodimer to DNA. Hopewell and Ziff
(1995) recently reported proliferation of the nerve growth factor
responsive PC12 cell line in the absence of a functional Max. Whether
c-Myc can induce apoptosis in a Max-independent manner in these cells,
however, is not yet known.
Here we have studied the effect of dominant-negative mutants of c-Myc and Max on proliferation. Max mutants with a mutated or deleted basic region efficiently blocked serum-induced DNA synthesis. Unexpectedly, c-Myc mutants expressing only the BR/HLH/LZ or HLH/LZ domain rapidly induced apoptosis.
Figure 1:
Schematic
survey of c-Myc and Max mutants used in this work. Wild-type c-Myc and
Max proteins are shown (stippled box). Dimerization domains
are indicated by arrows. The mutant MaxCKII has replaced Ser-11 by Ala in a known CKII phosphorylation site.
MaxBRmut has replaced amino acids Glu-32 and Lys-34 by Ile and Glu in
the DNA-binding domain, respectively. MaxBR
has
deleted the DNA-binding domain (positions 1-35). The mutant ctMyc
has deleted the amino-terminal region up to the basic domain at
position 354, and ctMycBR
has additionally deleted 13
amino acids with the basic region. MycHLH and MycLZ consist of either
the HLH domain (amino acids 368-409) or the LZ domain (amino
acids 410-439) of c-Myc. NLS indicates the nuclear localization
signal M1 of c-Myc, the cross-hatched boxes indicate the viral epitopes
recognized by the 12CA5 or C3 antibody. For details of construction of
all mutants see ``Materials and
Methods.''
A similar mode of action is expected for a
dominant-negative c-Myc mutant lacking the transcriptional activation
domain and BR (ctMycBR). Overexpression of
ctMycBR
should sequester Max protein, which is then
no longer available for dimerization with wild-type c-Myc. A second
c-Myc mutant, ctMyc, differs from the previous mutant by having an
intact BR. Heterodimers of ctMyc
Max have lost their
transactivation potential but should be able to bind specifically to
DNA.
To ensure nuclear transport of all mutant c-Myc proteins, the
NLS M1 of c-Myc (Dang and Lee, 1988), which maps outside of the
BR/HLH/LZ region, was cloned 3` of the LZ. The Max mutants contain
their NLS at the authentic position in the carboxyl terminus. All
mutant proteins were tagged with epitopes of viral proteins of
influenza virus (MaxBR, MaxBRmut, MycHLH, MycLZ) or
polio virus (ctMycBR
, ctMyc) in order to detect them
by indirect immunofluorescence staining. For unknown reasons, the polio
epitope did not work satisfactorily. Instead, we used the antibody 9E10
(Evan et al., 1985), which recognizes the LZ domain of c-Myc.
A survey of all mutants used in this work is shown in Fig. 1.
Unprogrammed reticulocyte
lysate extract (translation reaction without addition of exogenous RNA)
already revealed a binding activity (Fig. 2A, lane
2, marked with an asterisk). This shift was competed by
oligonucleotides CM1 containing the CACGTG motif (Blackwell et
al., 1990) and CM1mut with an inversion of the middle CG of the
E-box motif (Fig. 2A, lanes 4 and 5),
indicating that this activity is not specific for the CACGTG-binding
motif. Additionally, this complex could not be disrupted or
supershifted by the addition of antibodies directed against Max (Fig. 2B, lane 2) and c-Myc (data not shown)
and therefore contains no MaxMax homodimers or c-Myc
Max
heterodimers. The amount of this endogenous shift activity varied
considerably depending on the batch of the reticulocyte lysate.
Figure 2:
Inhibition of sequence-specific DNA
binding of ctMycMax heterodimers by dominant-negative mutants.
Binding reactions contained in vitro translated proteins as
indicated, the radiolabeled oligonucleotide CM1 (Blackwell et
al., 1990) with the consensus c-Myc-binding site, and binding mix. A, Lanes 2-9, 1.5 µl of the indicated
lysate was incubated at 37 °C for 10 min; lanes
10-12, 0.75 µl of ctMyc lysate was mixed with 0.75
µl of Max lysate and incubated at 37 °C for 10 min. ctMyc and
Max generate a specific gel shift (arrow). Labeled CM1
oligonucleotide alone (lane 1) and unprogrammed reticulocyte
lysate (UL) (lane 2) served as controls. The
endogenous unspecific complex in the unprogrammed lysate is marked with
an asterisk. Competition experiments were performed by
addition of a 200-fold molar excess of unlabeled oligonucleotide CM1 (200xCM1) or CM1mut (200xCM1mut). B, lanes 1, 3, and 5, 1.5 µl of ctMyc
lysate (lane 1) or 0.75 µl of ctMyc lysate mixed with 0.75
µl of Max lysate (lanes 3 and 5) were incubated
at 37 °C for 10 min. Lane 2, 1 µl of
-Max
antibody and 1.5 µl of ctMyc lysate were mixed and incubated at 37
°C for 10 min. Lanes 4 and 7, 1 µl
-Max
antibody (lane 4) or 1 µl of antibody 12CA5 (lane
7) were mixed with 0.75 µl of Max lysate and incubated at 37
°C for 5 min. Subsequently, 0.75 µl of ctMyc lysate was added,
and incubation at 37 °C was continued for 5 min. Lanes 6 and 8, 1 µl of Myc antibody 9E10 (lane 6) or
antibody Ki67 (lane 8) were mixed with 0.75 µl of ctMyc
lysate and incubated at 37 °C for 5 min. Subsequently, 0.75 µl
of Max lysate was added, and incubation at 37 °C was continued for
5 min. ctMyc
Max shifts are supershifted by
-Max antibodies (open arrows), whereas the
-Myc (9E10) (data not shown)
and
-tag (12CA5) antibodies inhibit formation of ctMyc
Max
specific shifts (filled arrow). In lanes 5-8 another batch of reticulocyte lysate was used as in lanes
1-4. C, lane 1, 0.75 µl of ctMyc
lysate was mixed with 0.75 µl of Max lysate and incubated at 37
°C for 10 min. Lanes 2 and 3, 0.75 µl of
ctMyc lysate was mixed with 2 µl of MaxBR
lysate (lane 2) or MaxBRmut lysate (lane 3) and incubated at
37 °C for 5 min. Subsequently, 0.75 µl of Max lysate was added,
and incubation was continued for 5 min. Lanes 4-6, 0.75
µl of Max lysate was mixed with 2 µl of MycLZ lysate (lane
4), MycHLH lysate (lane 5), or ctMycBR
lysate (lane 6) and incubated at 37 °C for 5 min.
Subsequently, 0.75 µl of ctMyc lysate was added, and incubation at
37 °C was continued for 5 min. MaxBR
, MaxBRmut,
and ctMycBR
but not MycHLH and MycLZ compete for the
formation of ctMyc
Max-specific gel shifts. For further details,
see ``Materials and Methods.''
When
ctMyc was added to the binding reaction, an additional shift appeared
that most likely is produced by a heterodimer of ctMyc and endogenous
Max, which is already present in the extract (Fig. 2A, lane 3). This shift can be competed with the oligonucleotide
CM1 but not with CM1mut (Fig. 2A, lanes 4 and 5). We observed that the Max protein alone did not produce a
specific shift (Fig. 2A, lane 6). This was not
unexpected since phosphorylation of Max at an amino-terminal casein
kinase II (CKII) site in the reticulocyte lysate inhibits DNA-binding
of MaxMax homodimers but not c-Myc
Max heterodimers
(Berberich and Cole, 1992). We could confirm this observation by using
the mutant MaxCKII
, which has replaced Ser-11 by Ala (Fig. 2A, lanes 7-9). Mixing of ctMyc
and Max generated a strong shift that can be explained by synergistic
action of both proteins (Fig. 2A, lanes
10-12).
The specificity of the observed shifts was also
analyzed by using antibodies. The -Max antibodies reduced and/or
supershifted ctMyc
Max complexes (Fig. 2B, lanes 1-4). Antibodies specific for the c-Myc LZ domain
or the viral tag of Max only inhibited formation of ctMyc
Max
specific shifts (lanes 6 and 7).
We next tested
whether the presence of the mutant Max and c-Myc proteins in the
binding reaction can compete for the formation of the ctMycMax
shift. Addition of a 2-fold excess of MaxBR
,
MaxBRmut, or ctMycBR
protein to the binding reaction
resulted in a clear reduction of the ctMyc
Max-specific shift (Fig. 2C, lanes 2, 3, and 6), whereas addition of unprogrammed reticulocyte lysate (data
not shown) or MycHLH and MycLZ had no effect (Fig. 2C, lanes 4 and 5). In summary,
ctMycBR
, MaxBR
, and MaxBRmut were
able to disrupt a ctMyc
Max complex in vitro and
therefore act in a dominant-negative manner as expected.
Figure 3:
Dominant-negative mutants of Max inhibit
DNA synthesis in serum-stimulated cells. a, time scheme of the
proliferation assay. b, quiescent 3T3-L1 fibroblasts were
microinjected with expression plasmids encoding
MaxBR, MaxBRmut, or
-galactosidase. 20 h after
serum stimulation, the cells were fixed and analyzed by
immunocytochemistry. DNA synthesis of cells expressing the Max mutants
or
-galactosidase (these cells are marked by white
arrows) was measured by incorporation of BrdUrd. c,
quantitative evaluation of at least five experiments. Each experiment
was done with more than 200 cells expressing either
MaxBR
, MaxBRmut, or
-galactosidase, and the rate
of BrdUrd incorporation was determined. BrdUrd incorporation in
serum-starved cells was consistently less than 3%, in serum-stimulated
cells more than 70%.
Expression
of MaxBR and MaxBRmut differed in regard to their
cellular distribution. MaxBRmut was almost exclusively demonstrable in
the nucleus, whereas MaxBR
was also observed in the
cytoplasm in a considerable portion of cells. As a control, an
expression plasmid coding for
-galactosidase was injected.
Expression of
-galactosidase had no significant effect on
induction of DNA synthesis (Fig. 3b). A quantification
of the proliferation assay is shown in Fig. 3c.
Figure 4:
Apoptotic cell expressing
ctMycBR, which shows the characteristic cytoplasmic
blebbing and chromatin condensation. Cells were fixed 6 h after
microinjection, ctMycBR
was detected by
immunofluorescence with the
-Myc antibody (9E10), and chromatin
was stained with 4,6-diamidino-2-phenylindole (DAPI). The
apoptotic cell is indicated by an arrow.
We performed kinetic experiments to determine the
onset of apoptosis in cells expressing ctMyc and
ctMycBR. For this purpose, the injected cells were
inspected and photographed in intervals of 2 h after microinjection (Fig. 5). Expression of mutant c-Myc proteins could be detected
in the nucleus and in the cytoplasm 2 h after microinjection (data not
shown). The rate of expressing cells was >70% of injected cells and
thus in the range as observed for other proteins
(MaxBR
, MaxBRmut,
-galactosidase). At this time,
cells expressing ctMyc and ctMycBR
still showed a
normal shape. However, after 4 h, the cells changed their morphology,
and bright spots became apparent in the field of injected cells. The
number of affected cells consistently increased between 6 to 10 h,
while at the same time many of the affected cells disintegrated. After
12 h, cells with a normal shape expressing ctMyc or ctMycBR
were no longer detectable. Apoptosis was not observed in cells
expressing
-galactosidase, MaxBRmut (see Fig. 3), and
MaxBR
(Fig. 5).
Figure 5:
Kinetics of induction of apoptosis by
ctMyc and ctMycBR. Quiescent 3T3-L1 fibroblasts were
microinjected with expression plasmids coding for ctMyc,
ctMycBR
or MaxBR
, and the field of
injected cells was photographed in intervals of 2 h (1, 2, 3, 4, 5, 6) .
After 12 h, cells were fixed, ctMyc and ctMycBR
were
detected by immunofluorescence staining with the
-Myc antibody
(9E10), and MaxBR
was detected with the antibody
12CA5(7) . Chromatin was stained with
4,6-diamidino-2-phenylindole(8) .
Dominant-negative mutants of c-Myc have been successfully used in earlier studies to inhibit transformation (Dang et al., 1989; Sawyers et al., 1992) and induction of DNA-synthesis (Hermeking et al., 1994). These c-Myc mutants carried deletions in the transactivation domain between amino acids 40 and 178 and therefore differ markedly from the mutants employed in this study, which have deleted the amino terminus up to amino acids 354 and 367. Notably, the mutant with the deletion in the transactivation domain did not induce apoptosis in quiescent 3T3-L1 fibroblasts in microinjection experiments (Hermeking et al., 1994; data not shown).
Induction of apoptosis by wild-type c-Myc in
serum-starved mouse fibroblasts can be inhibited by refeeding the cells
with medium containing high serum levels (Evan et al., 1992).
Therefore, similar kinetic experiments as carried out with
serum-starved 3T3-L1 fibroblasts were performed with proliferating
3T3-L1 cells in the presence of 10% FCS. Since proliferating
fibroblasts showed a high mobility on the surface of the coverslip with
a permanent change of positions, a similar documentation of the results
as shown for quiescent fibroblasts in Fig. 5was impossible.
Despite this problem, plasmids coding for ctMycBR and
ctMyc were microinjected in proliferating cells. Both mutants showed a
normal expression rate of 70% after 2 h. Similar to quiescent cells,
many of the ctMycBR
and ctMyc expressing cells showed
an apoptotic morphology after 6-8 h. 12 h after microinjection,
cells with a normal morphology expressing the c-Myc mutants were no
longer detectable (data not shown).
Figure 6:
Co-expression of MaxBR inhibits apoptosis induced by ctMyc and
ctMycBR
. Expression plasmids were mixed in a ratio of
2:1 (300 ng/µl MaxBR
, 150 ng/µl ctMyc or
ctMycBR
) and microinjected in quiescent 3T3-L1
fibroblasts. Cells were fixed after 12 h, and ctMyc and
ctMycBR
were detected by immunofluorescence staining
with the
-Myc antibody (9E10). Chromatin was stained with
4,6-diamidino-2-phenylindole.
Figure 7: Separate expression of MycHLH and MycLZ does not induce apoptosis. Quiescent 3T3-L1 fibroblasts were microinjected with expression plasmids coding for MycHLH or MycLZ, fixed after 12 h, and the mutants were detected by immunofluorescence staining with the antibody 12CA5. Chromatin was stained with 4,6-diamidino-2-phenylindole.
Expression of the c-myc gene in serum-starved mouse
fibroblasts induces DNA synthesis and at the same time triggers
apoptosis. Both events have been shown to require heterodimerization of
c-Myc and Max. Here we have studied dominant-negative mutants
inhibiting specifically DNA binding of c-MycMax heterodimers. The
phenotypes of c-Myc and Max mutants after expression in 3T3-L1 mouse
fibroblasts differed markedly. While Max mutants inhibited
serum-induced DNA synthesis, c-Myc mutants rapidly induced apoptosis.
The
mutant ctMycBR inhibited binding of the
ctMyc
Max heterodimer to the cognate E-box to a similar extent as
the mutants MaxBRmut and MaxBR
did. From this
observation, we expected an inhibitory effect on DNA synthesis also for
the mutant ctMycBR
. However, this mutant induced
apoptosis in serum-stimulated cells before the onset of DNA synthesis
could be measured. Apoptosis was induced by this mutant at low and high
serum levels and also observed after expression of ctMyc.
Induction
of apoptosis by wild-type c-Myc requires dimerization of the HLH/LZ
domains of c-Myc and Max (Amati et al. 1993b). The following
reasons argue against dimerization of Max and the c-Myc mutants as
requirement for induction of apoptosis. (i) The presence of Max does
not confer DNA binding activity to the
ctMycBRMax heterodimer. Alternatively,
ctMycBR
may act by sequestering Max from other DNA
binding complexes. However, Max is also sequestered by
MaxBR
and MaxBRmut, which do not induce apoptosis.
(ii) Instead, the mutants MaxBR
and MaxBRmut suppress
ctMycBR
-induced apoptosis, indicating that complexes
of ctMycBR
MaxBR
and
ctMycBR
MaxBRmut are unable to induce apoptosis.
We therefore conclude that ctMycBR
and ctMyc interact
probably with other factor(s) than Max to induce apoptosis in 3T3-L1
cells.
Induction of apoptosis by
wild-type c-Myc depends on the presence of the amino-terminal
transcriptional activation domain (Evan et al., 1992) and
dimerization of c-Myc with Max (Amati et al., 1993b). This
suggests specific gene-regulatory activity of the c-MycMax
complex during induction of apoptosis. In this respect, induction of
apoptosis by the c-Myc mutants differs markedly from wild-type
c-Myc-induced apoptosis. It is very unlikely that the c-Myc mutants can
still regulate target genes of wild-type c-Myc. However, since the
mutants ctMycBR
and ctMyc probably can still interact
with other transcription factors, both mutants may also induce
apoptosis by altering gene expression. The changes in gene expression
are probably severe and induce apoptosis even in the presence of high
serum. Although we are far from understanding how the mutants ctMyc and
ctMycBR
act, the presented data are of importance for
our understanding of c-Myc protein function.
A c-Myc mutant with a deleted transactivation domain (amino acids 40-178) is unable to induce apoptosis in quiescent 3T3-L1 fibroblasts but has been shown to inhibit serum-induced DNA synthesis (Hermeking et al., 1994). This implicates that the central part of the c-Myc protein (amino acids 179-354) either by itself or by binding of other proteins, controls the interaction of cellular proteins with the carboxyl-terminal HLH/LZ domain of c-Myc. Deletion of the central part of the c-Myc protein renders the HLH/LZ domain highly promiscuous for otherwise tightly controlled interactions with cellular factors. For this reason, c-Myc mutants with a deleted transactivation-domain are more useful as dominant-negative mutants than mutants consisting only of the HLH/LZ domain (Mukherjee et al., 1992; Hermeking et al., 1994).