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Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3900-3912
Posttranslational Regulation of Myc Function in Response to Phorbol
Ester/Interferon- -Induced Differentiation of v-Myc-Transformed
U-937 Monoblasts
By
Fuad Bahram,
Siqin Wu,
Fredrik Öberg,
Bernhard Lüscher, and
Lars-Gunnar Larsson
From the Department of Genetics and Pathology, University of Uppsala,
University Hospital, Uppsala, Sweden; and the Institut für
Molekularbiologie, Medizinische Hochschule Hannover, Carl-Neuberg
Strasse 1, Hannover, Germany.
 |
ABSTRACT |
The transcription factors of the Myc/Max/Mad network are important
regulators of cell growth, differentiation, and apoptosis and are
frequently involved in tumor development. Constitutive expression of
v-Myc blocks phorbol ester (TPA)-induced differentiation of human U-937 monoblasts. However, costimulation with interferon- (IFN-) and TPA restores terminal differentiation and G1
cell-cycle arrest despite continuous expression of v-Myc. The mechanism
by which TPA + IFN- counteract v-Myc activity has not been
unravelled. Our results show that TPA + IFN- treatment led to an
inhibition of v-Myc- and c-Myc-dependent transcription, and a
specific reduction of v-Myc:Max complexes and associated DNA-binding
activity, whereas the steady state level of the v-Myc protein was only
marginally affected. In contrast, TPA + IFN- costimulation neither
increased the expression of Mad1 or other mad/mnt family genes
nor altered heterodimerization or DNA-binding activity of Mad1. The
reduced amount of v-Myc:Max heterodimers in response to treatment was accompanied by partial dephosphorylation of v-Myc and c-Myc.
Phosphatase treatment of Myc:Max complexes lead to their dissociation,
thus mimicking the effect of TPA + IFN-. In addition to modulation of the expression of Myc/Max/Mad network proteins, posttranslational negative regulation of Myc by external signals may, therefore, be an
alternative biologically important level of control with potential
therapeutic relevance for hematopoietic and other tumors with
deregulated Myc expression.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE MYC FAMILY of proto-oncogenes
plays an important role in the regulation of cell proliferation,
differentiation, and apoptosis. c-myc expression is normally
tightly linked to the proliferative state of cells and its product is
suggested to be one of the key nuclear transmitters of mitogenic
signals. Constitutive c-Myc expression results in enforced cell-cycle
progression in the absence of mitogens, blocks differentiation of
various cell types, and may induce apoptosis if survival factors are
lacking.1 Chromosomal aberrations involving myc
family loci have been implicated in the generation of a variety of
tumors in vertebrates, in particular within the hematopoietic system
and are often strongly correlated to a poor prognosis.2
Myc proteins are transcription factors of the basic region
(b)/helix-loop-helix (HLH)/leucine zipper (Zip)-family. The HLHZip motif of Myc mediates dimerization with the bHLHZip protein
Max,3-5 an interaction that seems necessary for the
biological activities of c-Myc.6-9 The basic regions enable
Myc:Max heterodimers to bind specifically to a subclass of E-box DNA
elements,10-12 whereas the N-terminal transactivation
domain of c-Myc mediates stimulation of E box-driven
promoters.6,7,13-15 Suggested target genes of c-Myc/Max
complexes include  -prothymosine, ODC, cdc25A,
eIF-2, eIF-4E, CAD, and MrDb.16
In addition to the interaction with c-Myc, Max has recently been shown
to form heterodimers with a number of other bHLHZip proteins, including
Mad1, Mxi1, Mad3, Mad4, of the Mad family,17-19 and
Mnt/Rox.20,21 These heterodimers bind to the same E-box sequence as Myc:Max complexes, and Mad/Mnt may, therefore, compete with
Myc for binding to Max and/or to DNA. The Mad/Mnt proteins have been
shown to inhibit cell growth22-24 and to repress
transactivation and transformation by Myc.17,19,20,22,25-28
These activities require an interaction with the repressor protein
mSin3 and associated proteins including N-CoR/SMRT and the histone
deacetylases HDAC1/HDAC2,29 suggesting that Mad/Mnt repress
transcription at least, in part, through remodeling of chromatin.
The mad genes seem to be expressed primarily in differentiated,
nonproliferative tissues.18,19,28,30-34 These observations have lead to the hypothesis that the Myc/Max/Mad network may constitute a molecular switch in which the prevalence of Myc-containing versus Mad-containing heterodimers determines whether cells enter a
differentiation pathway or remain in a proliferative, undifferentiated
state. This view has been further supported by recent observations that ectopic expression of Mad1 promotes differentiation of murine erythroleukemia cells,35 whereas targeted disruption of
mad1 inhibits cell-cycle exit and delays terminal
differentiation of granulocytic precursor cells.36
mxi1-deficient mice exhibit abnormalities in the homeostasis of
several differentiated organs and increased cancer
susceptibility.37
It is generally believed that the main regulation of the Myc/Max/Mad
network occurs at the level of the expression of its components.
Although posttranslational regulation in response to cellular signaling
has been described for many other transcription factors,38
this has not been clearly established for the Myc/Max/Mad proteins, and
therefore remains an open question. With the U-937 monocytic
differentiation model we have previously shown that interferon-
(IFN-), if combined with classical inducers of differentiation such
as the phorbol ester TPA, restores differentiation and G1 cell-cycle arrest in v-Myc-transformed U-937 cells, despite a continuous expression and nuclear localization of v-Myc.39
The mechanism by which TPA + IFN- costimulation counteracts the
activity of Myc has not been unravelled. We hypothesized that this
treatment could either increase the expression or activity of members
of the mad-family and, thereby, restore the balance within the
Myc/Max/Mad network or directly inhibit the activity or downregulate
the steady state level of v-Myc.
Our results suggest that TPA + IFN- costimulation does not increase
the expression of mad-family genes or the activity of Mad1, but
interferes with Myc function as demonstrated by inhibition of the
transactivating and DNA-binding ability of v-Myc and c-Myc. This likely
occurs through the observed destabilization of Myc:Max heterodimers, a
mechanism that is correlated with modification of Myc by
dephosphorylation. Therefore, our findings provide evidence for an
alternative signal-mediated pathway to interfere with Myc function in
addition to the Mad/Mnt family of proteins, which potentially may have
therapeutical relevance for hematopoietic and other tumors with
deregulated c-Myc expression.
| |
MATERIALS AND METHODS |
Cell culture and differentiation assays.
U-937 human histiocytic lymphoma cells40 were cultured in
RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) and
antibiotics. BK3A chicken bursal lymphoma cells41 were
grown in Dulbecco's modified Eagle's medium containing 5% FCS, 1%
chicken serum, and 10% tryptase broth. The U-937 clones U-937-myc-6
(expressing the OK10 v-myc gene), U-937-neo-6 (lacking the
v-myc gene), the parental clone U-937-GTB, and the U-937-1
clone have been described previously.31,42 Exponentially
growing cells (105/mL) were induced to differentiate in
medium containing 1.6 × 10 8 mol/L TPA (Sigma, St
Louis, MO) and/or 100 U/mL IFN- (generously provided by Dr G.R.
Adolf, Ernst-Boehringer Institute, Vienna, Austria). Differentiation
was defined by the expression of mature monocytic antigens; the
adhesion molecules (-integrins) CD11a/LFA-1, CD11b/Mac1, and
CD11c/p150.95, and the receptor for monomeric IgG, FcRI. Also CD4,
which is expressed on immature U-937 cells and downregulated on
differentiation, was analyzed. 3H-thymidine incorporation
and the immunofluorescence studies were performed as described
previously.39 For the analysis of CD4, CD11c, CD11a, CD11b,
and FcRI the monoclonal antibodies (MoAbs) OKT4 and LeuM5 (Becton
Dickinson, Mountain View, CA), MHM24, 44, and 32.2 (from the IVth
Leukocyte Typing Workshop collection43) were used respectively.
Immunoprecipitations and Western blot analysis.
For 35S and 32P in vivo cell labeling 5 × 106 cells were labeled for 40 minutes in 1 mL of
methionine-free RPMI-1640 containing 0.15 mCi of
35S-methionine or for 2 hours in 1 mL of phosphate-free
RPMI-1640 containing 0.35 mCi of 32P-orthophosphate,
respectively. For high and low stringency precipitations, cells were
lysed in AB and Tris lysis buffer, respectively33 and
immunoprecipitated with specific antibodies. An equal number of
TCA-precipitable counts (for 35S-labeled proteins) or
aliquots of lysates with equal amounts of protein (unlabeled proteins)
were used for each sample. The washing procedure for high and low
stringency immunoprecipitations has been described.33 The
samples were analyzed on 10% to 15% sodium dodecyl
sulfate-polyacrylamide (SDS-PAGE) gels.
For phosphatase treatment, L-buffer lysates of 35S-labeled
cells were immunoprecipitated with antibodies whereafter the
immunocomplexes were washed in L-buffer and AP buffer (100 mmol/L Tris
pH 8, 50 mmol/L MgCl2, 1% aprotinin) before treatment with
5 U alkaline phosphatase (Sigma) at 30°C for 20 minutes either in the
presence or absence of 100 mmol/L  -glycerophosphate. The
immunocomplexes were then extracted twice with 200 µL L-buffer and
the proteins analyzed by SDS-PAGE.
The immunoblot and immunostaining procedure were performed as described
previously.33 The blots were developed by enhanced chemiluminescence (Amersham Pharmacia Biotech, Uppsala, Sweden) using
horseradish peroxidase (HRP) conjugated anti-rabbit Ig (Amersham) for
rabbit antisera or streptavidin conjugated HRP (Amersham) for
biotinylated antibodies.
For detection of Myc, IG-13, a rabbit pan-Myc antiserum generated by
immunizing with a bacterially produced full-length human c-Myc protein
(I. Guzhova, L.-G. Larsson, unpublished data, April 1993),
5042, a rabbit antiserum specific for chicken v- and c-Myc (kindly
provided by Dr S. Hann and Dr R.N. Eisenman) and C-33 monoclonal
pan-Myc antibodies (Santa Cruz Biotechnology [SCB], Santa Cruz, CA) were used. The C-33 MoAb was conjugated with 10 µg
biotin-X-NHS (Calbiochem-Novabiochem, La Jolla, CA) per milligram antibody in 0.1 mol/L NaHCO3 pH 7.4, 0.1 mol/L NaCl for 1 hour at room temperature (RT) followed by dialysis in
phosphate-buffered saline (PBS). For analysis of Mad1 and Max, 266-4 rabbit Mad1 antiserum,33 C-19 rabbit Mad1 antiserum (SCB),
91-4 rabbit Max antiserum,33 and C-17 rabbit Max antiserum
(SCB) were employed.
Transfections and assays for reporter activity.
For electroporation, 2 × 107 cells in 0.5 mL of RPMI-1640
were mixed with the DNA in a 0.4 cm electroporation cuvette (Bio-Rad, Laboratories, Richmond, VA), incubated for 5 minutes at RT and electroporated at 960 µF, 300 V in a Bio-Rad gene pulser. After 10 minutes on ice, the cells were suspended in RPMI-1640, 10% FCS at a
concentration 0.3 × 106 cells/mL and cultured for 16 to
24 hours. For differentiation experiments, the transfected cells were
split into aliquots, which were treated with the differentiation agents
as described above. The harvested cells were washed in PBS, suspended
in 1 mL of TEN (40 mmol/L Tris pH 7.5, 1 mmol/L ethylenediamine
tetraacetate (EDTA), 150 mmol/L NaCl) and left on ice for 10 minutes.
After centrifugation, the cells were resuspended in 200 µL of 0.25 mol/L Tris pH 7.8, lysed by freeze thawing, and then the lysate was clarified by centrifugation. For CAT-assays an equal amount of protein
per sample was incubated with 1 µCi 14C-chloramphenicol
(Sigma), 0.2 mg/mL butyryl-CoA (Sigma) in 100 µL at 37°C for 1 hour. After extraction with mixed xylenes (Sigma) the amount of
acetylated 14C-labeled chloramphenicol was determined by
scintillation counting. For luciferase assays, cells were lysed in a
buffer containing 25 mmol/L Tris pH 7.8, 2 mmol/L EDTA, 10% glycerol,
1% Triton X-100, and 2 mmol/L DTT for 10 minutes on ice. Extracts were
mixed with assay buffer (25 mmol/L glycyl glycine, 15 mmol/L
MgSO4, 5 mmol/L adenosine triphosphate), then 50 µL of 25 µmol/L luciferin was injected and the light emission was measured for
3 seconds by using a Lumat LB 9501/16 Berthold luminometer
(EG&G Berthold, Bad Wildbad, Germany). For -gal assays extracts were
mixed with 300 µL of Z buffer (60 mmol/L
Na2HPO4, 40 mmol/L
NaH2PO4, 10 mmol/L KCl, 1 mmol/L
MgSO4) after which 100 µL of O-nitrophenyl
-D-galactopyranoside (ONPG; Sigma) (4 mg/mL in Z buffer) was added.
The solution was then incubated at 37°C until the yellow color
appeared after which the reaction was stopped with 250 µL of 1 mol/L
Na2CO3. The absorbance was measured at 414 nm
in a Titertek Multiscan MCC/340.
The following DNA constructs were used in the transient transfections:
PrT-CAT contains a 3.1 kb fragment including 250 bp of the
-prothymosine promoter, exon 1, intron 1, and part of exon 2 of the
-prothymosine gene linked to a CAT reporter gene. The E-box situated
in the first intron is replaced by two Gal4 binding sites in the
GalmE-PrT-CAT construct.44 The two constructs were kindly
provided by Dr M. Eilers. m4mintk-Luc and mintk-Luc contains a minimal
tk promoter ( 32/+51) in front of a luciferase reporter gene with or
without a tetramer of the CMD oligonucleotide upstream of the tk
promoter.45 pCMV-myc contains a full length c-myc
cDNA downstream of the cytomegalovirus (CMV)-promoter in the construct
pEQ176P2. In pCMV-Myc BR, the c-myc cDNA insert of
pSPMycBR,7 lacking the basic region, was cloned into the CMV expression vector pCB6. CMV-Luc contains the luciferase gene driven
by a CMV promoter. hubactp/lacZ was used to normalize the transfection
efficiency and contains 4 kb of the human -actin promoter linked to
a lacZ reporter gene,46 kindly provided by Dr U. Lendahl.
For stable transfections, 35 µg of linearized m4mintk-Luc DNA was
cotransfected with 5 µg of pRSV-hygr, which was used as a
selectable marker. Hygromycin (400 µg/mL) (Calbiochem Inc, San Diego,
CA) was added 2 days after transfection and resistant clones were
picked 3 weeks after transfection, expanded, and analyzed for the
presence of the integrated construct by luciferase assays and
polymerase chain reaction analysis.
DNA-binding assays.
The solid phase DNA-binding assay (SODA) was performed as described
previously.33 Briefly, cells were lysed under low
stringency conditions in L-buffer and equal amounts of protein were
immunoprecipitated with antibody. The washed samples were incubated
with 1 ng of 32P-labeled oligonucleotide in 30 µL of
gelshift buffer containing 100 ng of salmon sperm DNA for 25 minutes at
room temperature.33 After washing the amount of bound
oligonucleotide was measured in a scintillation counter. The
oligonucleotides used were CMD (5'-TCAGACCACGTGGTCGGG),
which contains an optimal Myc/Max binding site (underlined) and CMM
(5'-TCAGACCAGCTGGTCGGG) containing a mutated Myc/Max
binding site (underlined).
Two-dimensional gel electrophoresis.
35S-labeled proteins were immunoprecipitated, collected,
and washed as described above and incubated in 50 µL of 20 mmol/L
Tris pH 8, 1 mmol/L EDTA, 0.3% SDS, 1% DTT at 95°C for 10 minutes
and then 50 µL of 2D-sample buffer (9 mol/L Urea, 2%
2-mercaptoethanol, 2% Pharmalyte 3-10, 0.5% Triton X-100, BFB) was
added. The samples were separated in the first dimension by isoelectric
focusing with immobiline drystrips pH 3-10 (Pharmacia Biotech, Uppsala, Sweden) in a horizontal Multiphor II system (Pharmacia Biotech) followed by SDS-PAGE in the second dimension. For blocking experiments, the IG-13 pan-Myc antiserum was incubated with 10 µg of recombinant c-Myc protein for 30 minutes at RT. Phosphatase treatment was performed
by incubating the washed immunoprecipitates with 150 U of
 -phosphatase (Biolabs, Beverly, MA) for 30 minutes at 30°C.
| |
RESULTS |
TPA + IFN- costimulation restores differentiation and growth
arrest in v-Myc expressing U-937 human monoblasts.
Growth and differentiation of v-Myc-expressing U-937-myc-6 and
parental U-937-GTB cells in response to TPA, IFN-, or the combination of TPA + IFN- were determined by analysis of
3H-TdR-incorporation and the expression of a number of
differentiation-related surface antigens, respectively (Fig
1). The incorporation of 3H-TdR
declined during the first day of TPA-induced differentiation in both
cell lines, but proliferation was gradually regained the following days
in the v-Myc-expressing cells (Fig 1A). However, TPA + IFN-
costimulation prevented the late increase in
3H-TdR-incorporation in agreement with our previous
report.39 IFN- alone reduced DNA synthesis with slower
kinetics.
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| Fig 1.
Growth and differentiation of v-Myc expressing
U-937-myc-6 and parental U-937-GTB cells. Cells were induced by TPA,
IFN-, or TPA + IFN- and analyzed at the indicated time points.
(A) 3H-TdR incorporation. Cells were labeled with 10 µCi
of 3H-TdR for 1 hour. (B) CD11a, (C) CD11b, (D) CD4, (E)
CD11c, and (F) FcRI expression. The surface antigen expression was
measured by fluorescence-activated cell sorter analysis by using
specific antibodies.
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In contrast to the U-937-GTB cells, TPA-induced changes in the
expression of the early differentiation markers CD11a, CD11b, and CD4
occurred only transiently in U-937-myc-6 cells, whereas the later
markers CD11c and FcRI were completely inhibited, indicating that
the differentiation process was initiated normally but was subsequently
aborted in parallel with resumed cell growth (Fig 1B through F).
Costimulation with TPA + IFN- restored the expected changes in the
expression of all five differentiation markers analyzed. IFN- alone
influenced transiently the expression of some of the surface antigens,
whereas others were unaffected.
In summary, TPA-induced differentiation and growth arrest, although
initiated normally, was aborted in the U-937-myc-6 cells but was
restored by TPA + IFN- costimulation. Because v-Myc is continuously
expressed in these cells39 (see below), this result raises
the question how TPA + IFN- costimulation might override the
Myc-induced block of differentiation.
v-Myc is continuously synthesized and the expression of Mad1 is not
restored in TPA + IFN--treated U-937-myc-6 cells.
To investigate the synthesis of Mad1, c-Myc and v-Myc, and Max, the
respective proteins were immunoprecipitated from
35S-labeled U-937-myc-6 and U-937-GTB cell extracts with
specific antibodies. In agreement with previous reports, the synthesis of Mad1 increased in response to TPA in U-937-GTB
cells30,31,33 but not in U-937-myc-6 cells (Fig
2A), indicating that the continued presence
of v-Myc directly or indirectly influenced the expression of Mad1. The
synthesis of v-Myc increased somewhat in the U-937-myc-6 cells, whereas
the c-Myc synthesis was reduced after TPA stimulation in both cell
lines. No major TPA-induced changes were observed in the expression of
the p21 and p22 Max proteins. From these findings, one possibility is
that TPA + IFN- might re-establish differentiation in v-Myc
expressing U-937 cells by restoring the expression of Mad1 or by
increasing the expression of other members of the Mad/Mnt family.
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| Fig 2.
Myc, Mad, and Max protein synthesis in U-937-myc-6 and
U-937-GTB cells. (A) U-937-myc-6 and U-937-GTB cells were treated with
TPA for 3 days before 35S-methionine labeling. High
stringency lysates were immunoprecipitated with pan-Myc (IG-13), Mad1
(C-19), and Max (C17) antibodies and analyzed by SDS-PAGE. (B) Time
course of Myc, Mad1, and Max proteins synthesis during stimulation of
U-937-myc-6 cells. TPA, IFN-, or TPA + IFN- were added for the
time indicated and the proteins were analyzed as in (A).
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A detailed kinetic study showed that the synthesis of Mad1 increased
during the first 4 hours in TPA- and TPA + IFN--treated cells (Fig
2B), but in contrast to the U-937-GTB cells (Fig 2A), returned to basal
levels by 12 hours after both treatments. Northern blot analysis of
mad1 mRNA showed a similar pattern of expression (data not
shown). The expression of mxi1 mRNA, which normally increases
late after TPA treatment in U-937 cells,31 remained unchanged after TPA, IFN-, or TPA + IFN- treatment of U-937-myc-6 cells (data not shown). Furthermore, costimulation with IFN- did not
increase the expression of mad3, mad4 or mnt
mRNA (data not shown). v-Myc was continuously synthesized, whereas
synthesis of the endogenous c-Myc protein gradually decreased after TPA and TPA + IFN- treatment, but was less affected by IFN- alone (Fig 2B). The different treatments induced some transient changes in
the synthesis ratio of the two Max proteins p21 and p22. Thus, we
concluded that terminal differentiation induced by TPA + IFN- cannot
be explained by a restored expression of Mad1 or by an increased mRNA
expression of other mad/mnt-family genes.
TPA + IFN- inhibits Myc-regulated
E-box-dependent reporter-gene activity.
Because TPA + IFN- treatment neither reduced the synthesis of v-Myc
nor increased the expression of Mad1 or other mad/mnt-family members we hypothesized that TPA + IFN- might affect the activity of
Myc or Mad through post-translational mechanisms or alternatively bypass the Myc/Max/Mad network by affecting their downstream targets. We addressed this question by using the Myc-regulated -prothymosine promoter and an artificial Myc-responsive promoter to determine whether
the transactivating capacity of Myc could be modulated in response to
TPA + IFN-. The Myc-responsive promoter/reporter gene
constructs44,45,47 are depicted in Fig
3A.
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| Fig 3.
Regulation of E-box-dependent promoter/reporter activity
during induced differentiation of U-937 cells. (A) Schematic
presentation of the reporter constructs. A detailed description of the
constructs is given in Materials and Methods. (B) Repression of basal
E-box-dependent PrT-CAT activity by a dominant negative c-Myc mutant.
20 × 106 cells of the indicated U-937 clones were
electroporated with 20 µg of Pr-T-CAT or GalmE-PrT-CAT with or
without 30 µg of pCMV-myc BR. CAT activity was determined 16 hours
after transfection. CAT or luciferase activities (B through D) were
normalized to -gal activity by cotransfection with 20 µg
hubactp/lacZ. (C) PrT-CAT activity during differentiation of U-937
clones. PrT-CAT or GalmE-PrT-CAT were electroporated with or without 5 µg of pCMV-myc as described in (B). Cells from separate
electroporations were pooled and divided into aliquots, which were
treated with the inducers indicated. (D) m4mintk-Luc activity during
differentiation of U-937 clones. The experiment was performed as
in (C). As a reference, mintk-Luc, lacking E-boxes and pCMV-Luc, which
contains the luciferase gene driven by a CMV-promoter, was used. (E)
Kinetics of m4mintk-Luc activity during differentiation of U-937-m112
cells. U-937-m112 is a subclone of U-937-myc-6 containing a stably
integrated m4mintk-Luc construct. The cells were induced by TPA,
IFN-, or the TPA + IFN- for the indicated time points and
assayed for luciferase activity. The results (C through E) are
presented as percentage of untreated U-937-myc-6 cells. (B-E) The data
of at least three independent experiments are summarized.
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Electroporation of the -prothymosine promoter constructs PrT-CAT and
GalmE-PrT-CAT (the latter lacking the Myc-responsive E-box) into three
U-937 clones showed that the PrT-CAT activity was somewhat higher in
the v-Myc-expressing U-937-myc-6 cells than in the U-937-1 or
U-937-neo-6 control cells and that the activity of GalmE-PrT-CAT was
reduced to 40% of that of PrT-CAT (Fig 3B). To estimate the
contribution of Myc in these transactivations, a dominant negative
c-Myc mutant lacking the basic region (pCMV-mycBR) was
cotransfected. In all three cell lines, this resulted in repression of
the basal activity of PrT-CAT to a level that corresponded to the level
of activity obtained with GalmE-PrT-CAT (Fig 3B). Cotransfection of
pCMV-mycBR had little effect on GalmE-PrT-CAT. These results
indicate that Myc is the main contributor of E-box-dependent transcription of the -prothymosine promoter in U-937 cells, which is
in agreement with the conclusion drawn previously by using other cell
types.44
Treatment of U-937-neo-6 cells lacking v-myc with TPA, IFN-
and TPA + IFN- all reduced PrT-CAT activity, but had little effect
on GalmE-PrT-CAT (Fig 3C). In U-937-myc-6 cells, TPA did not reduce the
activity of PrT-CAT to the same extent as in U-937-neo-6 cells, but
IFN- and in particular TPA + IFN- were efficient inhibitors. To
further establish that TPA + IFN- inhibited the transactivating
properties of c-Myc, PrT-CAT, and GalmE-PrT-CAT were cotransfected with
a c-Myc-expression vector. pCMV-myc stimulated PrT-CAT activity 3 to
4-fold, but did not affect GalmE-PrT-CAT. IFN- alone and in
particular TPA + IFN- inhibited pCMV-myc-induced reporter activity,
whereas TPA alone resulted in little inhibition (Fig 3C). GalmE-PrT-CAT
was unaffected under these conditions.
Similar results were obtained with an additional Myc-inducible
promoter/reporter construct, m4mintk-Luc, driven by four copies of a
Myc:Max E-box binding site upstream of a minimal tk promoter. The
m4mintk-Luc reporter activity was 2 to 3-fold higher in U-937-myc-6 cells as compared with U-937-neo-6 cells (Fig 3D), presumably because
of their constitutive expression of v-Myc, whereas the activity of the
mintk-Luc construct, lacking the E-boxes, was low. TPA, IFN-, and
TPA + IFN- all reduced the m4mintk-Luc activity in U-937-neo-6 to
30% to 40% of untreated cells. In contrast, the luciferase activity
was unaffected or even increased in TPA-treated U-937-myc-6 cells,
whereas both IFN- and TPA + IFN- reduced the activity
to 30% of untreated cells (Fig 3D). The activity of mintk-Luc was not
affected under these conditions. It is relevant to point out that in
addition to Myc other E-box-binding transcription factors may
contribute to the activity of m4mintk-Luc. To rule out that the
treatments led to a general inhibition of transactivation, the activity
of a CMV-promoter/reporter construct (pCMV-Luc) was assayed. The
activity of pCMV-Luc (Fig 3D) and other promoters, such as a Rous
Sarcoma Virus long terminal repeat and a long herpes simplex virus
tk-promoter (data not shown) were rather enhanced by TPA and TPA + IFN- treatment and were not affected by IFN- alone.
To assess, whether the treatments would affect m4mintk-Luc activity in
a chromatin environment, a U-937-myc-6 clone with stable integration of
the m4mintk-Luc construct (U-937-m112) was used. Kinetic experiments
showed that TPA treatment led to a reduced reporter activity during the
first day of treatment, which increased again and had even exceeded
that of untreated cells at 72 hours. In contrast, TPA + IFN-
treatment led to a permanent inhibition of the reporter activity, which
was down to 20% of control values by 24 hours. IFN- alone inhibited
the reporter activity with a somewhat slower kinetic and to a lesser
extent as the costimulation with TPA. Taken together these experiments
suggest that TPA + IFN-, in particular, but also IFN- treatment
alone inhibits transactivation by v-Myc and c-Myc.
TPA + IFN- costimulation decreases the
DNA-binding activity of Myc.
Several alternative mechanistic explanations can be envisioned for the
ability of TPA + IFN- and IFN- to inhibit v-Myc and c-Myc
activity as measured by transactivation assays. One possible explanation is that Myc is competed out from its DNA-binding sites by
Mad/Mnt-family proteins or other E-box binding transcription factors or
alternatively that TPA + IFN- directly regulate the function of the
transactivation, DNA binding or heterodimerization domains of Myc.
Because the activity of a c-Myc-transactivation domain/Gal4 fusion
protein did not seem to be affected by TPA + IFN- in transient
transactivation assays using reporter constructs driven by Gal4 DNA
binding sites (data not shown), we measured the capacity of native Myc,
Max and Mad1-complexes to bind specifically to DNA in response to TPA + IFN- treatment. For this purpose, we used SODA (solid-phase
DNA-binding assay), which we recently developed based on partial
purification of native Myc/Max/Mad complexes.33
Unfortunately, the conventional electrophoretic mobility shift assay
can not be applied for these types of studies as previously
discussed.33
Low stringency immunoprecipitations of Myc-, Mad1-, and Max-complexes
from v-Myc-expressing U-937 and control (U-937-GTB and U-937-neo-6)
cells were performed by using antisera that do not interfere with
dimerization or DNA binding. The immunocomplexes were incubated with
labeled oligonucleotides containing a Myc/Max binding site (CMD) or a
mutated version of CMM as described.33 Figure
4A shows that -Max or -Myc
immunocomplexes bound specifically to the CMD oligonucleotide, because
the binding was competed with unlabeled CMD but not CMM
oligonucleotides and only background levels were detected with the
preimmune serum or labeled CMM. The DNA-binding activity of -Myc
immunoprecipitates decreased after TPA-induced differentiation of
U-937-GTB and U-937-neo-6 control clones, but remained high in v-Myc
expressing U-937-myc-6 cells. Smaller variations in DNA binding were
seen in the -Max immunoprecipitations. The DNA binding of Mad1
immunocomplexes was low in untreated cells but increased after
TPA-stimulation of U-937-GTB but not in U-937-myc-6 cells. The
DNA-binding activity measured seemed to correlate with the expression
levels of the respective proteins.
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| Fig 4.
Analysis of the DNA-binding activity of Myc, Mad1, and
Max complexes during induced differentiation of U-937 cells. (A)
Lysates from untreated U-937-myc-6, U-937-neo-6, and U-937-GTB cells or
cells induced by TPA for 3 days were subjected to SODA.33
The lysates were immunoprecipitated under low stringency conditions by
using specific antisera and incubated with 32P-labeled CMD
oligonucleotide, containing a Myc/Mac binding site or a mutated version
(CMM) after which the amount of bound oligonucleotide was measured. The
specificity was determined by competition with an excess of cold CMD or
CMM oligonucleotide and by using preimmune (PI) serum. (B) Kinetic
study of the DNA binding of total Myc-, v-Myc-, and total
Max-containing and Mad1-containing complexes during differentiation of
U-937-myc-6 cells. The cells were induced with TPA, IFN-, or the TPA + IFN- for the indicated time and subjected to SODA as in (A). The
antisera used in (A) and (B) were IG-13 (anti-pan-Myc), 5042 (anti-chicken Myc), 266-4 (anti-Mad1) or 91-4 (anti-Max). The results
are presented as a percentage of untreated cells. The error bars
represent the standard deviations of the means from at least three
independent experiments.
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|
A kinetic analysis of the DNA binding in response to TPA, TPA + IFN-, and IFN- treatment in U-937-myc-6 cells using pan-Myc antiserum showed that the DNA-binding of total v-Myc + c-Myc
immunocomplexes rapidly declined after TPA treatment but increased
again at 24 hours, reaching 75% of the value of untreated cells by 72 hours (Fig 4B). In TPA + IFN--treated cells the total Myc DNA
binding continued to decline past 4 hours and reached a minimum by 24 hours and thereafter. IFN- alone also reduced Myc DNA binding but to
a lesser extent. Experiments using a v-Myc-specific antiserum showed
that the v-Myc DNA-binding activity did not change substantially early
after TPA stimulation but increased at later time points. In TPA + IFN--treated cells, however, the v-Myc DNA binding gradually decreased with the lowest values at 24 and 72 hours. The v-Myc DNA
binding was to a lesser extent reduced also after treatment with
IFN- alone.
Only slight variations in the DNA-binding of -Max immunocomplexes
were observed in response to any of the treatments (Fig 4B). These
complexes, which contain at least Myc and Mad1 (Fig 5B and ref 33), presumably represent the
sum of all different Max-containing heterodimers as well as of Max:Max
homodimers. In contrast, the DNA binding of the Mad1-containing
immunocomplexes increased transiently within 1 hour after TPA treatment
with maximal activity 4 hours after stimulation but returned to control
levels at late time points (Fig 4B). IFN- and TPA + IFN-
treatment for 72 hours did not increase the binding above the basal
level. In summary, the inhibition of Myc-regulated, E-box-dependent
transactivation is paralleled by a decrease in Myc-specific DNA binding
in response to TPA + IFN- treatment in U-937-myc-6 cells.
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| Fig 5.
Western blot analysis of total Myc and Myc:Max complexes
during induced differentiation of U-937-myc-6 cells. (A) Early kinetics
of TPA- and TPA + IFN--stimulation for the times indicated. (B)
Late kinetics of TPA- and/or IFN--stimulation. Upper panels:
analysis of total Myc expression. (A) v- plus c-Myc and (B) v-Myc were
immunoprecipitated from low stringency lysates by rabbit pan-Myc
antibodies (IG-13) or rabbit chicken Myc-specific antibodies (5042),
respectively, followed by Western analysis using monoclonal pan-Myc
antibodies (C-33). Middle and lower panels: analysis of Myc:Max
complexes and Max expression, respectively. Aliquots of the same
lysates as in the upper panels were immunoprecipitated by using C17 Max
antibodies followed by Western analysis of coimmunoprecipitated Myc
(middle panel) and of Max (lower panel). Equal amounts of protein were
immunoprecipitated for each lane. The figure shows representative
results from 1 of 4 independent experiments.
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TPA + IFN- costimulation reduces the amount of
Myc:Max complexes.
The results above suggest that the inhibition of Myc activity by TPA + IFN- is at least, in part, because of an effect on the DNA-binding
capacity of Myc itself rather than increased competition from Mad1 or
other E-box-binding transcription factors at the Myc/Max binding site
or interference with the transactivation domain of Myc. The reduced
DNA-binding activity of Myc-containing complexes could be due to a
reduced steady state level of v-Myc, to an inhibition of Myc DNA
binding per se, or to reduced Myc:Max complex formation or stability.
To address these questions we measured the steady state levels of
v-Myc, c-Myc, and Max, and the capacity of Myc and Mad1 to
heterodimerize with Max in response to treatment.
Coimmunoprecipitation studies were performed under low stringency
conditions followed by Western blot analysis of coimmunoprecipitated proteins. The total amount of v-Myc, c-Myc, and Max immunoprecipitated with chicken Myc-specific, pan-Myc, and Max antiserum, respectively, were compared with the amounts of Myc coimmunoprecipitated with Max
antiserum (Fig 5). The amount of v-Myc:Max complexes (middle panel) and
the total level of v-Myc (upper panel) was relatively stable during the
first 4 hours of TPA and TPA + IFN- stimulation (Fig 5A). Thereafter
the amount of v-Myc:Max complexes declined significantly after TPA + IFN- treatment reaching a minimum at 24 hours (six-fold reduction),
whereas only a slight reduction in total v-Myc steady state level was
observed (Fig 5B). IFN- alone also reduced the amount of v-Myc:Max
as compared with total v-Myc. The reduction of v-Myc:Max complexes in
response to TPA + IFN- or IFN- treatments was not caused by
decreased steady state levels of Max as shown in the lower panels of
Figs 5A and B. In contrast, both the steady state level of v-Myc and
the amount of v-Myc:Max complexes increased 2 to 2.5-fold after 72 hours of TPA treatment. The steady state levels of c-Myc declined after TPA + IFN- or TPA treatment but not significantly after IFN- treatment as expected from the synthesis rates of c-Myc (Figs 2 and 5;
data not shown). Few c-Myc:Max complexes were observed in TPA + IFN-
and IFN- treated cells as compared with TPA-treated cells at 72 hours post induction (Fig 5B, middle panel). In conclusion, these
results suggest that a potentially important consequence of the
treatment with TPA + IFN- and with IFN- alone is the reduction in
the fraction of total v-Myc and c-Myc, which is complexed with Max.
The amount of Max coimmunoprecipitated with Mad1 increased transiently
after 1 to 4 hours of TPA induction, but then returned to basal levels
(Fig 6), essentially reflecting the
synthesis of Mad1 (Fig 2). No increase in the amount of Max in complex
with Mad1 was observed late after TPA + IFN- treatment in agreement with the unchanged synthesis of Mad1. These findings argue against an
increase in Mad1 protein due to stabilization or increased Mad1:Max
affinity. The apparent increase in Max and Mad1:Max after IFN-
treatment was because of overloading.
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| Fig 6.
Western blot analysis of total Max and of Mad1:Max
complexes during induced differentiation of U-937-myc-6 cells.
U-937-myc-6 cells were induced with TPA, IFN-, or TPA + IFN-
for the times indicated. (Upper panel) Max was coimmunoprecipitated
from low stringency lysates using C19 Mad1 antibodies followed by
Western blot analysis of Max by using C17 Max antibodies (Lower panel).
Max was immunoprecipitated from aliquots of the same lysates with C17
Max antibodies followed by immunoblot analysis using the same
antibodies.
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TPA + IFN- costimulation results in modification
of Myc.
The data presented above suggest that the inhibition of Myc-induced
transactivation and of the DNA-binding activity of Myc by TPA + IFN-
is because of reduced Myc:Max heterodimerization. Destabilization of
Myc:Max heterodimers could be the result of TPA + IFN--induced
unidentified proteins competing for Myc or Max, or direct
modification(s) of Myc or Max by for instance
phosphorylation/dephosphorylation.
To assess whether the overall level of phosphorylation of v-Myc and
c-Myc changed in response to TPA + IFN- treatment in U-937-myc-6 and
U-937-GTB cells, aliquots of untreated and induced cultures were in
vivo labeled with 35S-methionine and
32P-orthophosphate in parallel, whereafter the Myc proteins
were immunoprecipitated and analyzed by SDS-PAGE. Fig
7A shows that the relative intensity of the
32P-labeled v-Myc and c-Myc proteins were significantly
reduced as compared to the corresponding 35S-labeled
proteins in TPA + IFN- treated, but not in untreated or TPA-treated
cells, indicating dephosphorylation of the Myc proteins in response to
the former treatment. To substantiate this further we analyzed
immunoprecipitated 35S-labeled Myc proteins from untreated
and treated cells by isoelectric focusing followed by SDS-PAGE (Fig
7B-G). In response to TPA + IFN- v-Myc (long arrow) shifted towards
the basic side as compared to marker spots (short arrows) (Fig 7C). To
a lesser extent, IFN- alone also induced a shift toward a more basic
pI (data not shown), whereas TPA did not (Fig 7E). A shift toward a
basic isoelectric position might be caused by dephosphorylation.
Therefore, we treated immunoprecipitated Myc from control and TPA + IFN--treated cells with phosphatase and analyzed the products on 2D
gels. The different Myc isoforms collapsed into one major species that
migrated at a pI similar to the most basic isoform after TPA + IFN-
treatment (Fig 7F and G). Together these findings suggest that Myc
proteins are dephosphorylated on stimulation with TPA + IFN-.
Because of the moderate level of Myc in these cells we have,
unfortunately, not been able to obtain enough
32P-incorporation into the proteins to perform 2D
phosphopeptide analysis to establish which phosphorylation sites are
affected in response to TPA + IFN--stimulation. Presently, we are
investigating whether costimulation with IFN- affects Myc function
in a similar way in other cell types in which such studies may be
easier to perform.
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| Fig 7.
TPA + IFN- costimulation induces modification of
Myc. (A) Aliquots of untreated or stimulated U-937-myc-6 and U-937-GTB
cells were in vivo labeled with 32P-orthophosphate (upper
panel) or 35S-methionine (lower panel) as indicated
whereafter cell lysates were immunoprecipitated with IG-13 pan-Myc
antiserum. (B through G) 35S-labeled lysates of untreated
(B and F), TPA + IFN-- (C, D, and G) or TPA stimulated (E)
U-937-myc-6 cells were immunoprecipitated with IG-13 pan-Myc antibodies
and analyzed by 2D-gel electrophoresis as described in Materials and
Methods. In (D) the antibody was blocked by incubation with recombinant
c-Myc protein. In (F) and (G) the immunoprecipitates were phosphatase
treated. The positions of v-Myc and c-Myc and of two unspecific spots
are indicated by long and short arrows, respectively. (H) BK3A cells
were labeled with 35S-methionine, lysed in L-buffer and
Myc:Max complexes immunoprecipitated using Myc-specific antibodies
(5042). Treatment with alkaline phosphatase (PPase) in the presence or
absence of -glycerophosphate (-GP) was performed as indicated.
Max extracted in L-buffer (S) or bound to Myc (P) was analyzed by
SDS-PAGE and fluorography.
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We hypothesized that this dephosphorylation might be the basis for the
reduced interaction with Max and, thus, be the trigger for the
inhibition of Myc-specific DNA binding and transactivation. To evaluate
whether dephosphorylation might affect Myc:Max dimer stability, low
stringency Myc immunoprecipitates of 35S-labeled cell
lysates were phosphatase treated and the amount of Max that was either
bound to Myc or free determined. After phosphatase treatment, Max had
an increased mobility (because of dephosphorylation of C-terminal
sites; B.L., unpublished data, October 1994) and a substantial portion
was in the supernatant (Fig 7H). No release from Myc was observed after
mock incubation or when the activity of the phosphatase was blocked.
Although the dephosphorylation of Myc observed in vivo is only partial and no dephosphorylation of Max was seen (data not shown), these findings are consistent with the results discussed above;
dephosphorylation of Myc in Myc:Max heterodimers leads to a decreased
stability of the complex.
| |
DISCUSSION |
Constitutive expression of v-Myc blocks induced differentiation of
human U-937 monoblasts similar to other cellular differentiation systems.42,48-53 However, costimulation with IFN- and
TPA restores terminal differentiation and G1 cell-cycle
arrest despite continuous expression of v-Myc39; Figs 1 and
2). These findings suggest that the combination of TPA with IFN- can
interfere with the growth promoting and differentiation blocking
activities of Myc. We envisioned that these signals could counteract
Myc by at least three different mechanisms. First, by increased
expression or activity of members of the mad-family. Second, by
acting on Myc directly through inhibition of its activity or through
affecting its steady state level by a posttranslational mechanism.
Third, by acting downstream of Myc; for instance, through independent regulation of Myc target genes.
Our results show that the induced expression of Mad1 and the subsequent
increased formation of Mad1:Max heterodimers and associated DNA-binding
activity observed normally during TPA-induced differentiation of U-937
cells30,31,33 occurred only transiently in v-Myc expressing
cells (Figs 2, 4, and 6). This indicates a direct or indirect role of
Myc in the regulation of the mad1 gene. Restored differentiation by TPA + IFN- was, however, not accompanied by an
elevated level of Mad1 nor of its heterodimers with Max or its
DNA-binding activity (Figs 2, 4, and 6). Furthermore, costimulation with IFN- did not increase the expression of mxi1,
mad3, mad4 or mnt mRNA (data not shown).
Together, the results argue against a model were TPA + IFN- restore
differentiation and growth arrest by using the Mad/Mnt family to
counteract Myc activity. Apparently, terminal differentiation of U-937
cells can proceed with only basal level of mad-family gene
expression under these conditions. However, we cannot exclude
post-transcriptional and/or posttranslational regulation of Mad/Mnt
proteins, which potentially could contribute to restoring
differentiation on TPA + IFN- treatment. It is also possible that Mad1 could play a role during the early phase of TPA- and
TPA + IFN--induced differentiation and growth inhibition, during
which Mad1 is transiently induced.
Although we have no evidence supporting an involvement of the
mad/mnt-family, our results suggest that TPA + IFN--induced signals restore differentiation and growth arrest by interfering directly with the activity of Myc proteins. We base this conclusion on
three observations: (1) The Myc-regulated, E-box-dependent activity of
two different promoter/reporter gene constructs is inhibited by TPA + IFN-, in particular, but also by IFN- alone (Figs 3C-E); (2) The
DNA-binding activity of v-Myc and of total Myc (v-Myc + c-Myc) is
reduced after TPA + IFN- treatment and to a lesser extent by IFN-
treatment (Fig 4); (3) The fraction of total v-Myc, which interacts
with Max, declines in response to TPA + IFN- and IFN- treatment
(Fig 5).
The kinetics and the magnitude of the reduction in v-Myc:Max
heterodimers in response to TPA + IFN- is similar to the kinetics and the magnitude of reduction in DNA-binding activity of Myc and of
the inhibition of E-box-dependent promoter/reporter activity (Figs 3E,
4, and 5). Because DNA binding and transactivation by Myc are dependent
on heterodimerization with Max, it seems likely that the Myc:Max
interaction is the main target of the TPA + IFN--induced negative
signals. Reduced Myc:Max heterodimerization could potentially be the
result of increased competition for Max by other Mad/Mnt proteins than
Mad1. Because no antibodies of high enough quality directed against
these proteins were available, we were unable to address this question.
However, we find this explanatio |
TOP
ABSTRACT
INTRODUCTION