From the Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02912
Received for publication, October 11, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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The myc proto-oncogenes encode
transcriptional regulators whose inappropriate expression is correlated
with a wide array of human malignancies. Up-regulation of Myc enforces
growth, antagonizes cell cycle withdrawal and differentiation, and in
some situations promotes apoptosis. How these phenotypes are elicited
is not well understood, largely because we lack a clear picture of the
biologically relevant downstream effectors. We created a new biological
system for the optimal profiling of Myc target genes based on a set of isogenic c-myc knockout and conditional cell lines. The
ability to modulate Myc activity from essentially null to
supraphysiological resulted in a significantly increased and
reproducible yield of targets and revealed a large subset of genes that
respond optimally to Myc in its physiological range of expression. The
total extent of transcriptional changes that can be triggered by Myc is
remarkable and involves thousands of genes. Although the majority of
these effects are not direct, many of the indirect targets are likely to have important roles in mediating the elicited cellular phenotypes. Myc-activated functions are indicative of a physiological state geared
toward the rapid utilization of carbon sources, the biosynthesis of
precursors for macromolecular synthesis, and the accumulation of
cellular mass. In contrast, the majority of Myc-repressed genes are
involved in the interaction and communication of cells with their
external environment, and several are known to possess
antiproliferative or antimetastatic properties.
The Myc protein is a member of the basic
region/helix-loop-helix/leucine zipper (b/HLH/Zip) family of
transcriptional regulators and is capable of exerting both
transactivation and transrepression activities (1, 2). Transactivation
is mediated by binding as an obligate heterodimer with the b/HLH/Zip
factor Max to the consensus sequence CA(C/T)GTG (the E box) (3).
Transrepression is less well understood (4, 5). In either mode Myc is a weak transcriptional regulator, exerting most of its effects within the
2-5-fold range. In a general sense, the up-regulation of Myc strongly
enforces proliferation and growth, antagonizes cell cycle withdrawal
and differentiation, and in some situations promotes apoptosis (6-8).
In agreement, the down-regulation of Myc results in the attenuation of
both cell division and cell growth as well as protection against some
apoptotic processes (9-13). Despite extensive research, the specific
mechanisms by which these highly evident biological end points are
achieved are not well understood. This is largely because a
comprehensive list of biologically relevant Myc target genes has not
yet been defined.
A wide variety of techniques have been employed in the hunt for Myc
targets, ranging from differential expression screens, promoter
analysis, and informed guesswork (14-16) to the modern methods of
microarray profiling, serial analysis of gene expression, and
chromatin immunoprecipitation (17-23). This search has been complicated by several factors. First, the weak transcriptional effects
of Myc present significant experimental challenges. Second, by all
recent indications the total set of Myc targets may be very large.
Third, not all E boxes are bound by Myc, and transient transfection
studies do not adequately reflect regulation in a chromosomal context.
Fourth, comparing tumor cells expressing amplified Myc with
nonderegulated counterparts is complicated by the nonisogenic nature of
the cells. A widely used approach has been to compare cell lines
engineered to overexpress ectopic Myc with parental cells (17, 19, 21,
23). However, it is questionable to what extent this approach can
detect genes that respond optimally to physiological changes in Myc expression.
In an attempt to circumvent the latter problem, some time ago we
generated c-myc null cells that were derived by gene
targeting from an immortalized but otherwise nontransformed rat
fibroblast cell line (9). To date, the
c-myc To achieve maximum consistency in expression profiling we sought a
simple experimental regimen in which the only changing parameter was
the expression of c-Myc and in which a change in c-Myc status elicited
and clear and significant change in phenotype. We chose to use randomly
cycling, exponential phase cultures, and we developed conditions such
that cells experienced a constant environment and were in a balanced,
steady state of growth for significant periods of time. Under these
conditions c-myc null cells displayed a pronounced
phenotype, a 2-3-fold reduction in macromolecular synthesis
accompanied by a commensurate slowing of the cell cycle (9). Most
importantly, we showed that under these conditions both
c-myc+/+ and c-myc Expression profiling using a total of 81 Affymetrix GeneChip arrays was
performed in three experiments (Fig. 1).
First, we compared c-myc+/+ (TGR),
c-myc Cell Lines and Culture Conditions
TGR-1 is a hprt- subclone of the Rat-1 cell line
(27). HO15.19 (referred to as HO) is a homozygous c-myc null
derivative of TGR-1 generated by gene targeting (9). HOmyc3 was derived from HO15.19 by constitutively expressing murine c-myc
cDNA using a retroviral vector (10). HOmyc3 cells express c-Myc
protein at three to four times the level seen in TGR cells. HOMycER12 and HOMycER104 were derived in the same fashion to express MycER (25).
MycER is a hybrid protein consisting of the entire c-Myc polypeptide at
its N terminus and the ligand (estrogen) binding domain of the human
estrogen receptor at the C terminus. In the MycER construct used here
the estrogen binding domain has been mutated to be specific for the
agonist OHT. Retroviral vectors were packaged in BOSC cells (28), and
supernatants were used to infect HO15.19 cells. Colonies were selected
with 120 µg/ml hygromycin (Calbiochem), ring cloned, and expanded
into clonal cell lines. The mRNA encoding the MycER protein is thus
expressed constitutively from the viral long terminal repeat promoter,
and the activity of this promoter is not affected by OHT. OHT is
instead believed to elicit a conformational change in MycER which
allows the protein to become biologically active. All cultures were
grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% calf serum (Hyclone) at 37 °C in an atmosphere of 5% CO2, except BOSC cells, which were supplemented with 10%
fetal bovine serum (Hyclone). Great care was taken that cultures were cycling asynchronously and were in rapid and exponential phase of
growth (26). Briefly, cells were always split at subconfluent densities
(<50%) and at relatively low dilution (1:10 for
c-myc+/+ and 1:4 for
c-myc Molecular Biology Procedures
The c-myc, full-length MycER (25), and a deletion
mutant of MycER missing amino acids 106-143 (29) cDNAs were cloned
into the HpaI site of the pLXSH retroviral vector (30) using
standard procedures (31). Total RNA for Northern hybridization and
microarray analysis was isolated using TriZol reagent (Invitrogen).
Total RNA for quantitative real time PCR (qPCR) was isolated using the RNaqueous-4PCR kit (Ambion). Northern hybridization was performed using
the formaldehyde gel method, and 32P-labeled probes were
synthesized using the random oligonucleotide labeling method from
gel-purified restriction or PCR fragment templates as described
previously (32). qPCR was performed using the Applied Biosystems Prism
7700 Sequence Detector and software. Primers were designed using Primer
Express software (Applied Biosystems) for amplification of 100-bp
fragments. cDNA was generated using the TaqMan reverse
transcription kit and amplified using the SYBR green PCR and reverse
transcription PCR kits (Applied Biosystems). Amplification efficiencies
were determined by serial dilution of template cDNA for each gene.
All samples were run in triplicate. Glyceraldehyde phosphate
dehydrogenase (GAPDH) was used as the internal standard. GAPDH was used
because microarray profiling showed that the signals for six distinct
GAPDH probe sets were equivalent between TGR, HO, and HOmyc3 cell lines
under our exponential phase culture conditions. Protein samples were
prepared by lysing whole cells in radioimmune precipitation assay
buffer (33) supplemented with protease inhibitors. Immunoblotting was
performed as described previously (10, 34). The following antibodies
were used: c-Myc (Upstate Biotechnology, cat. 06-340), neomycin
phosphotransferase II, (5 Prime - 3 Prime, Inc., cat. 7-511721), and
actin (Sigma, cat. A5316). Horseradish peroxidase-conjugated secondary
antibodies were from Jackson Immunoresearch. Signals were visualized
using the ECL reagent (Amersham Biosciences).
Microarray Analysis (Fig. 1 Experiments)
Target cRNA was prepared and hybridized (45 °C, 16 h) to
GeneChip rat U34 arrays according to the manufacturer's directions (Affymetrix). Hybridized arrays were washed and stained using the
GeneChip fluidics station 400 and scanned using the Agilent GeneArray
scanner. Signals were analyzed using Microarray Suite 5.0 software
(Affymetrix). Data were normalized using a set target intensity of
1,500, published to a data base using MicroDB 3.0, and analyzed in Data
Mining Tool 3.0 software (Affymetrix). Analysis of each cell line
and/or condition was based on three biological replicates (RNAs
prepared from independent experiments performed at different times).
The replicates were used to calculate the means and standard deviations
for the expression values of all probe sets for each cell line and/or
condition. Probe sets were considered present if they received a
present call in two of the three biological replicates. Pairwise
comparisons between cell lines and/or conditions were made using
Student's t test (p < 0.05).
Analysis of Experiment 1
Myc-influenced expression patterns were assigned to four
categories: 1) activated by Myc; 2) repressed by Myc; 3) activated by
overexpression of Myc; and 4) repressed by overexpression of Myc (for
examples, see Fig. 4). Probe sets were categorized based on the
following criteria.
Category 1: Activated by Myc--
The ratio of the average
signal intensity between HOmyc3 and HO and/or between TGR and HO was
Category 2: Repressed by Myc--
The ratio of the average
signal intensity between HOmyc3 and HO and/or between TGR and HO was
Category 3: Activated by Myc Overexpression--
HO/Myc3 average
intensity values were significantly greater than those of TGR and those
of HO (p < 0.05), TGR and HO average intensity values
were not statistically different (p < 0.05), and the
ratio of average intensity values between TGR and HO was less than 1.4. 14 probe sets were moved from category 1 to category 3 based on visual
inspection (in these cases, the ratio of the average intensity values
between HO and HOmyc3 was greater than or equal to twice the ratio of
average intensity values between HO and TGR.
Category 4: Repressed by Myc Overexpression--
The HOMyc3
average intensity values were significantly less than those of TGR and
those of HO (p < 0.05), TGR and HO average intensity
values were not statistically different (p < 0.05), and the ratio of average intensity values between TGR and HO was greater than 0.7. Nine probe sets were moved from category 2 to category 4 based on visual inspection (in these cases, the ratio of
average intensity values between HO and HOmyc3 was less than or equal
to half the ratio of average intensity values between HO and TGR).
Analysis of Experiment 2
Probe sets were considered responsive to OHT if, at any given
time point, they displayed statistically significant (p < 0.05) differences between OHT and vehicle-treated replicates, and
the fold change between the means was Analysis of Experiment 3
Probe sets were considered to be direct Myc targets if
differences in expression between samples treated with cycloheximide plus OHT and those treated with cycloheximide alone at any time point
were statistically significant (p < 0.05) and had a
magnitude of
To assess the effect of OHT alone on RNA expression (in the absence of
the MycER transgene) TGR and HO cells were treated with OHT for 16 h (or vehicle for the same time period), and RNA was extracted and
subjected to microarray analysis. 288 of the total 8,799 probe sets on
the U94A chip were affected by OHT by a factor of Complementation of c-myc
As expected, HOmycER104 cells expressed higher levels of MycER protein
than HOmycER12 cells (Fig.
2A). Several assays were performed to examine the functionality of the
c-mycER transgene. Because c-Myc is known to
repress its own promoter, we examined the expression of the Neo
mRNA, which is encoded by one of the c-myc gene
targeting vectors and was placed under control of the endogenous
c-myc promoter by the homologous recombination event. Treatment of both HOmycER12 and HOmycER104 cells with OHT
clearly reduced the expression of the Neo mRNA (Fig.
2B). In contrast, repression of Neo by OHT was not observed
in the HO or HOmycER
We next examined the expression of several known c-Myc target genes in
HOmycER12 cells in the absence and presence of OHT (Fig.
2C). Activation of MycER resulted in the up-regulation of CAD mRNA and down-regulation of gadd45 mRNA, whereas the
expression of ornithine decarboxylase mRNA was essentially
unchanged or only weakly activated. The same behavior has been
documented for these genes by comparing TGR and HO cells under similar
exponential growth conditions (35). In control experiments, OHT had no
effect on the expression of CAD, gadd45, or ornithine decarboxylase
mRNAs in either TGR or HO cells (Fig. 2C, legend). We
therefore conclude that HOmycER12 cells in the absence of OHT resemble
c-myc
In preparation for experiments using OHT induction in the presence of
cycloheximide we examined the effect of cycloheximide on the expression
of the MycER protein (Fig. 2D). Expression of the MycER
protein declined rapidly and was barely detectable at 8 h after
cycloheximide and OHT addition. Because c-Myc protein is known to be
unstable, with a half-life estimated in the range of 30-40 min (36),
the turnover of the MycER protein after the addition of cycloheximide
was not unexpected. This observation unfortunately limits the utility
of cycloheximide as a tool to investigate the direct action of Myc to
targets that respond within a relatively short time frame.
Genes Differentially Expressed in c-myc Kinetic Analysis of c-Myc Target Gene
Regulation--
Exponentially cycling cultures of HOmycER12 cells were
treated with either OHT or vehicle (ethanol), and samples were
collected 2, 4, 8, and 16 h after treatment (experiment 2 in Fig.
1). The zero time point reference sample was harvested at the time of drug addition, resulting in 9 total RNA samples. The time course experiment was performed on three separate occasions to obtain three
biological replicates for a total of 27 RNA samples. Of the 611 probe
sets differentially expressed on the U34A chip in the TGR, HO, and
HOmyc3 comparison (experiment 1 in Fig. 1), 218 were responsive to OHT
in experiment 2. Because of some redundancy present on the chips, the
218 probe sets responsive to MycER correspond to 180 unique genes or
expressed sequence tag clusters. They were categorized further
according to their kinetics of induction as early, mid, or late
responding if the change in expression was first evident at 2-4 h,
8 h, or 16 h after the addition of OHT, respectively.
Finally, within these categories genes were grouped according to
general function (Table
I).
Representative induction profiles are shown in Fig.
5. The HOmycER104 cell line was also profiled in a time course of OHT induction with samples collected 0, 8, and 16 h after treatment. There was a high degree of concordance between the HOmycER12 and HOmycER104 data sets, thus providing additional verification (Table I).
Direct Targets of c-Myc--
Next, we sought to determine which of
the 180 unique genes and expressed sequence tags that we identified as
MycER-responsive may be direct targets. Exponentially cycling cultures
of HOmycER12 cells were treated with either OHT plus cycloheximide or
cycloheximide alone, and samples were collected 4, 8, and 16 h
after treatment (experiment 3 in Fig. 1). The zero time point reference
sample did not contain either drug. As previously, the time course
experiment was performed in three biological replicates. 21 of 180 OHT-responsive genes were designated as direct targets. Interestingly,
all 21 were in the Myc-activated category. In addition, we identified 24 activated genes and 16 repressed genes that appeared to be bona fide indirect targets. It is well documented that
cycloheximide alone can strongly influence gene expression. These
effects have the potential of significantly masking the influence of
Myc on the expression of bona fide target genes and
underscore the importance of doing cycloheximide only controls at all
time points. Indeed, all of the genes that failed the criteria of a
direct or indirect target showed significant induction or repression
resulting from cycloheximide alone. In these cases we do not believe
that a clear distinction between a direct and an indirect target can be
made. Representative plots of time courses in various categories are shown in Fig. 5, and the data for all 180 OHT-responsive genes are
summarized in Table I.
The three Affymetrix U34 GeneChips provide the most extensive
coverage of the rat genome available (26,261 probe sets, 20,691 unique
genes and expressed sequence tag clusters). The data set presented here
is thus the most comprehensive analysis of Myc-influenced gene
expression profiles to date. In total, we identified 1,527 probe sets
differentially expressed by 2-fold or more. Given that 43% of all U34
probe sets were expressed in Rat-1 fibroblasts, ~14% of the active
transcriptome is responsive to Myc within the 2-fold differential
expression cutoff. Because the U34 chips cover approximately half of
the rat genome, ~3,000 probe sets (~2,400 genes) can be estimated
to be Myc-responsive in this cell type. However, if the differential
expression criterion is relaxed to statistical significance only, the
Myc-responsive transcriptome becomes greater than 50% of all active genes.
The significantly increased yield of Myc-responsive genes achieved in
this study is the result primarily of our ability to modulate Myc
expression from almost zero to supraphysiological. This is clearly
evidenced by the fact that a previous report (17), which relied on
ectopic MycER expression in a normal (c-myc+/+)
cell background, identified only 36 MycER-responsive targets from a
total of 6,416 genes surveyed. Both studies used Affymetrix technology
and very similar criteria for data analysis. It is thus clear that
although a subset of Myc targets can respond to elevated Myc levels,
the great majority of responses occur in the range of physiological expression.
Given the widespread effects of Myc on gene expression, it is
noteworthy that only 36% of differentially expressed probe sets responded acutely to Myc activation in the HOMycER12 cell line. 22% of
the MycER-responsive genes have been identified in previous studies.
One limitation of the MycER activation regimen is the response time of
repressed genes because any observable effects depend on the turnover
of the preexisting mRNA. However, Myc-repressed genes comprised
45% of the OHT-responsive set and 51% of the total pool of
differentially expressed probe sets, indicating that the extent to
which Myc-repressed genes are being underestimated in the 16-h OHT time
course is likely to be minor.
The most likely explanation for the large fraction of
MycER-nonresponsive genes is that they represent longer term adaptive responses to the loss of c-Myc function. Although the existence of
direct targets with delayed responses cannot be ruled out, the fact
that many genes respond rapidly argues that factors other than Myc are
likely to account for the slow kinetics. Indeed, the list of
OHT-responsive genes includes many indirect targets, demonstrating that
even indirect effects can be rapid enough to score in the 16-h time
course. The morphological changes that accompany the addition of OHT to
HOmycER12 cells are slow, taking effect between 24 and
48 h. It is not unreasonable that the extensive adjustments of
cellular physiology which take place in response to loss and/or gain of
Myc activity would extend over one or even several cell cycles. It is
clear that the great majority of these changes are reversible because
95% of probe sets (on all three chips) that are differentially
expressed in c-myc Although the activation of MycER with OHT in the presence of
cycloheximide has been used frequently to differentiate direct and
indirect action of Myc, our data indicate that this method has limited
resolution. The major problems are the short life span of the MycER
protein and the presence of significant changes in gene expression
caused by cycloheximide alone. Of the 180 OHT-responsive genes, 119 (66%) were significantly affected by cycloheximide alone. Of the
remaining genes, 21 were activated and direct, 24 were activated and
indirect, and 16 were repressed and indirect. No directly repressed
genes were apparent. One possible explanation is that the resolution of
the cycloheximide methodology becomes critically limiting when the
short half-life of MycER and the effects of cycloheximide are
superimposed on the slow response of many repressed genes. Another
possibility is that repression by Myc depends on interaction with other
proteins, such as Miz-1, and that this interaction or the activity of
the interacting proteins is masked by cycloheximide artifacts.
Among Myc-activated genes, the relative proportion of direct and
indirect targets was approximately equal (21 and 24%, respectively). Because there are no obvious reasons why this relationship should not
also hold for genes subject to cycloheximide effects, by extrapolation we can expect ~47 directly activated targets among the 180 OHT-responsive genes. Because 38% of Myc-responsive probe sets were
found on the U34A chip, and the set of 3 chips covers approximately
half of the rat genome, we can expect ~247 directly activated Myc
targets in a rat fibroblast under exponential growth conditions.
Although our simple and easily controlled experimental design greatly
facilitates expression profiling, there are several reasons why it may
be underestimating the total spectrum of Myc-regulated genes. First, a
gene may not be affected equally by Myc under all growth conditions.
For example, the induction of Myc after serum stimulation of quiescent
cells could contribute significantly to the regulation of genes that
may respond only weakly under balanced, steady-state growth conditions.
Second, some genes may only be able to respond to Myc during a specific
segment of the cell cycle. Third, cell line- or cell type-specific
effects are also likely to be encountered. Fourth, some genes are
detected poorly or not at all by the current U34 probe sets
(e.g. p15 (Ink4b), p27 (Kip1), and Cdk7).
MycER-responsive genes identified in our profiling screen have diverse
functions (Table I). The largest single category on the Myc-activated
list (22 of 101 genes) are enzymes involved mostly in carbon
assimilation, anabolic pathways, and energy metabolism. Only 6 of these
have been reported previously (17, 21, 37, 38). What is striking is the
preponderance of enzymes that catalyze the first committed and
regulated steps of major pathways, such as glycolysis, biosynthesis of
purines, pyrimidines, polyamines, fatty acids, phospholipids,
S-adenosylmethionine (a key molecule in one carbon transfer
reactions), creatine (important, as creatine phosphate, in short term
energy storage), and NADPH (needed in most reductive anabolic
reactions). In addition to being rate-limiting and regulated
allosterically, many of the genes are regulated transcriptionally, and
their activation is correlated with rapid growth and proliferation.
Also prominent on the Myc-activated list are functions that positively
impact protein synthesis, including proteins involved in the synthesis
and processing of rRNA, the biogenesis of ribosomes, and translation
initiation and elongation factors. 17 genes fall in this category,
including RNA polymerase I, 8 of which have been reported previously.
The expression of 13 ribosomal proteins was decreased in
c-myc The appearance of several protein folding functions on the
Myc-activated list, both cytoplasmic and mitochondrial, is also consistent with an increased capacity for protein biosynthesis. Notable
among these are the mitochondrial chaperones and chaperonins prohibitin, BAP-37, Hsp60, Hsp10, and GrpE, all of which have been
identified previously as possible Myc targets. Interestingly, chaperones have been shown to have important roles in the control of
apoptosis. The GroEL/GroES homologs Hsp60/Hsp10 have proapoptotic effects involving caspase-3 activation (39, 40). In contrast, Hsp27 and
In a general sense, the majority of Myc-activated metabolic functions
are indicative of a physiological state geared toward the rapid
utilization of major carbon sources and the biosynthesis of precursors
for the synthesis of DNA, RNA, proteins, and lipids. Combined with the
up-regulation of the machinery for protein synthesis and folding these
changes would promote the accumulation of cellular mass, which is
required to support ongoing cellular proliferation. Myc also impacts
the expression of key G1 phase cell cycle regulators, raising the question as to which functions, metabolic or cell cycle,
are the primary effectors. The preponderance of genes that promote
metabolism and cell growth, as well as the documentation of increasing
numbers of bona fide direct Myc targets in this category,
makes it very unlikely that all these effects are secondary. Indeed,
preliminary evidence indicates that both metabolic and cell cycle
functions may be equally important: overexpression of an enzyme
involved in one carbon metabolism (serine hydroxymethyl transferase) or
a cell cycle regulator (Cdk4) both partially rescued the slow growth
phenotype of c-myc The Myc-repressed genes stand in stark contrast to the Myc-activated
targets: metabolic and protein synthesis functions are absent, and the
list is dominated by genes involved in cell adhesion, cell-cell
contact, extracellular matrix synthesis and modification, and vesicular
trafficking. In particular, the latter category has not been identified
previously as Myc-responsive. This list includes genes involved in
vesicular transport of secreted proteins, such as the calcium-binding
protein P22, Although much work will be needed to sort out direct and indirect
targets and to integrate fully the functions of the activated and
repressed genes, this study has significantly expanded our appreciation
of the impact of Myc on cellular physiology and has revealed a number
of intriguing novel candidates for drug targets. The total extent of
transcriptional changes that can be triggered in response to Myc
activity is remarkable, and it should be noted that many of the
indirect targets are likely to have important roles in mediating the
elicited cellular phenotypes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
cells have been used in two limited
profiling experiments that examined the expression of 4,400 rat (18)
and 6,355 mouse (24) cDNAs and expressed sequence tags in spotted
glass slide microarray formats. To create a new biological system for
the optimal profiling of Myc target genes, we have reconstituted
c-myc
/
cells with the conditionally active,
tamoxifen-specific c-Myc-estrogen receptor fusion protein
(MycER)1 (25). These new cell
lines allow the modulation of Myc activity from essentially null to supraphysiological.
/
cultures cycled uniformly, namely, that there were no cohorts of
differentially cycling or noncycling cells within a given culture (26).
/
(HO), and
c-myc
/
cells reconstituted with a
constitutive c-myc transgene (HOmyc3). This revealed the
total number of genes that respond to a sustained loss of c-Myc under
exponential growth conditions. Second,
c-myc
/
cells reconstituted with the
conditional c-mycER transgene (HOmycER) were
stimulated with 4-hydroxytamoxifen (OHT), and data were collected during a 16-h time course. This revealed the kinetics of the responses to Myc activation. Finally, the time course of induction with OHT was
performed in the presence of cycloheximide, revealing a subset of
direct transcriptional targets of c-Myc. All experiments, including the
growth of cells and preparation of RNA, were performed on three
separate occasions (independent biological replicates), and all data
were subjected to a statistical analysis of significance.
View larger version (27K):
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Fig. 1.
Schematic representation of expression
profiling experiments. The salient points of experimental design
are indicated as well as an overall summary of the obtained data. In
experiment 1 each RNA sample was used to interrogate 3 chips (U34A,
U34B, and U34C) for a total of 27 chips.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
cells). Cultures can thus be
maintained continuously at densities of 10-50% confluence (to avoid
any contact inhibition), and the relatively frequent passaging (every
3-4 days) and medium changes maintain a rapid growth rate. This
regimen was followed for a minimum of two passages before cells were
harvested for other experiments. MycER was activated with 200 nM OHT (Sigma). Dose-response studies showed that 200 nM OHT is saturating for the activation of MycER. OHT was
dissolved in absolute ethanol at 1 mM and stored at
80 °C. Mock-treated cultures received vehicle (ethanol) at a final
concentration of 0.02%. Protein synthesis was inhibited with 20 µg/ml cycloheximide (Sigma) which was added 30 min before the
addition of OHT. BrdUrd labeling and flow cytometry were performed as
described previously (9), except that the Vectastain Elite ABCTM and
NovaredTM kits (Vector Laboratories) were used for histochemical staining.
2.0.
0.50.
1.5. Of the 535 probe sets on
the U34A chip which were identified in the initial comparison of TGR,
HO and HOmyc3 cell lines (experiment 1), 142 probe sets were
OHT-responsive by the above criteria. An additional 460 probe sets
satisfied the OHT inducibility test (experiment 2, Fig. 1) but failed
the 2-fold induction limit set in the comparison of TGR, HO, and HOmyc3
cells (experiment 1). 76 probe sets were recovered from this list and
designated as c-Myc targets if the inducibility in the TGR, HO, and
HOmyc3 comparison (experiment 1) was
1.5. The resultant 218 (142 + 76) MycER-responsive probe sets out of the total 611 (535 + 76) probe sets represent 180 nonredundant genes. 75 of the 180 genes (41%) identified in HO/mycER12 cells satisfied the same
statistical criteria in HOmycER104 cells. Of the remaining 105 genes,
49 (27%) were already deregulated by the high basal Myc activity in
HOmycER104 cells, 36 (20%) behaved qualitatively similarly in
HOmycER104 cells but failed the t test, and 20 (11%) failed
the fold change test or behaved anomalously. Because the MycER protein
is capable of eliciting low Myc activity even in the absence of OHT, we
also asked whether this "leakiness" could mask potential responses
to Myc activation if, for example, probe sets were already maximally
induced/repressed before the addition of OHT. Probe sets were
considered leaky if the average intensity values in HOmycER12 cells
were statistically different (p < 0.05) and greater
(for Myc-activated genes) or smaller (for Myc-repressed genes) than the
average intensity values in HO cells. Probe sets that were leaky,
nonresponsive to OHT, unable to respond to elevated levels of Myc
(nonresponsive to OHT in HOmycER104 cells and/or not overexpressed in
HOmyc3 versus TGR cells), and expressed above a threshold
intensity value of 500 in TGR cells comprised less than 10% of the
probe sets identified in experiment 2.
1.5. Probe sets were classified as indirect targets of
Myc if differences in expression between samples treated with
cycloheximide alone were not statistically different (p < 0.05) from the untreated control and if differences in expression at
any time point between samples treated with cycloheximide plus OHT and
cycloheximide alone were not statistically significant
(p < 0.05).
2 in either TGR or
HO cells. 2 of the OHT-affected probe sets are on the list of 180 genes
reported in Table I. However, these probe sets were affected by OHT
only in HO cells and not in TGR cells. The genes affected by OHT alone
in HO cells are
-mannosidase II (M24353) and cytosolic
Na/K-transporting ATPase, B subunit (AA859920). The expression of these
genes was clearly affected in a comparison of TGR, HO, and HOmyc3 cells in the absence of OHT; however, because part of their response in
HOmycER cells may be the result the effect of OHT alone, further examination may reveal them to be indirect Myc targets.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
Cells with a
Conditional c-mycER Transgene--
The c-mycER
cDNA (25) was introduced into c-myc
/
cells using the retrovirus vector LXSH. Selection with hygromycin in
the absence of OHT resulted in an approximately equal mixture of slowly and rapidly growing colonies. Cells in the slow growing colonies displayed a flattened and spread-out morphology typical of
c-myc
/
cells, whereas cells in the rapidly
growing colonies had the fibroblastic morphology characteristic of the
parental c-myc+/+ cells. Expression of a
c-mycER transgene containing an internal deletion of Myc
sequences (MycER
106-143) did not generate any fast growing
colonies. The fast growing colonies are thus likely to be the result of
low levels of Myc activity elicited by the MycER transgene even in the
absence of OHT. Colonies of both types were picked and expanded into
cell lines. Two representative clones were chosen for further study:
HOmycER12, derived from a slow growing colony, and HOmycER104, derived
from a rapidly growing colony.
106-143 cell lines (Fig. 2B,
legend). Because expression of Myc accelerates the cell cycle and thus
increases the fraction of cells in S phase (9), we measured S phase
content of HOmycER12 and HOmycER104 cultures using BrdUrd labeling
(Fig. 3). The percentage of cells in S
phase was increased from 32 to 48% in HOmycER12 cells and from 42 to
57% in HOmycER104 cells after a 24-h treatment with OHT.
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Fig. 2.
Characterization of
c-myc /
cell lines reconstituted with the conditional c-mycER
transgene. A, expression levels of c-Myc and
MycER proteins. The indicated cell lines were harvested in the
exponential phase of growth and analyzed by immunoblotting (cell line
abbreviations: Myc3, HOmyc3; ER12, HOmycER12; ER104, HOmycER104; ER
,
HOmycER
106-143). Where indicated, OHT was included in the culture
medium for 16 h before harvest. The MycER
protein contains an
internal deletion (amino acids 106-143) and thus migrates faster than
MycER. Actin was used as the loading control. X-band, a
cross-reacting protein of unknown identity. B, repression of
knock-in Neo mRNA by MycER. HOmycER12 and HOmycER104 cells were
harvested in the exponential phase of growth and analyzed by qPCR.
Where indicated, OHT was included in the culture medium for 16 h
before harvest. GAPDH was used to normalize for equal input of RNA.
Repression of Neo mRNA by the 16-h OHT treatment was 1.7 ± 0.22-fold in the HOmycER12 cell line and 3.8 ± 1.1-fold in the
HOmycER104 cell line. To demonstrate that the effect of OHT on Neo
mRNA expression is dependent on MycER, HO, and HOmycER
106-143
cells were treated with OHT for 16 h (or propagated in parallel
without OHT), and RNA was harvested and analyzed by qPCR. All data were
normalized to GAPDH and are expressed relative to the no OHT condition,
which was set to a value of 1.0 for each cell line. The values in the
presence of OHT were: HO, 0.97 ± 0.17; HOmycER
106-143,
0.86 ± 0.11. C, effect of MycER on expression of
gadd45, CAD, and ornithine decarboxylase (ODC) mRNAs.
HOMycER12 cells were harvested in the exponential phase of growth and
analyzed by Northern hybridization. Where indicated, OHT was included
in the culture medium for 48 h before harvest. GAPDH was used as
the loading control. To demonstrate that the effect of OHT on gadd45,
CAD, and ornithine decarboxylase mRNA expression is dependent on
MycER, TGR and HO cells were treated with OHT for 48 h (or
propagated in parallel without OHT), and RNA was harvested and analyzed
by qPCR. All data were normalized to GAPDH and are expressed relative
to the no OHT condition, which was set to a value of 1.0 for each gene
and cell line. The values in the presence of OHT were as follows: CAD,
TGR 0.95 ± 0.20 and HO 1.14 ± 0.15; gadd45, TGR 1.07 ± 0.13 and HO 1.11 ± 0.12; ornithine decarboxylase, TGR
0.93 ± 0.13 and HO 1.45 ± 0.11. D, expression of
MycER protein in the presence of cycloheximide (CHX).
HOMycER12 cells in exponential phase of growth were treated with OHT
and cycloheximide. Note that cycloheximide was added 30 min before OHT.
Samples were harvested at the indicated times and analyzed by
immunoblotting. Actin was used as the loading control.
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Fig. 3.
Proliferation of
c-myc /
cell lines reconstituted with MycER. The fraction of cells in S
phase was determined using pulse labeling with BrdUrd. The indicated
cell lines were pulsed for 30 min during the exponential phase of
growth, and BrdUrd incorporation was visualized using in
situ immunocytochemical methods. Where indicated, OHT was included
in the culture medium for 24 h before harvest.
c-myc+/+ (TGR) and
c-myc
/
(HO) cells processed as above (in the
absence of OHT) were used as controls. OHT does not affect the BrdUrd
incorporation of TGR or HO cells (data not shown).
/
(HO) cells and that addition of OHT
elicits a c-myc+/+ (TGR)-like phenotype.
/
Cells--
Total RNA was extracted from exponentially cycling cultures
of TGR, HO, and HOmyc3 cells, and expression profiling was performed using Affymetrix U34 rat GeneChips. Each cell line was grown on three
separate occasions, and each of the corresponding RNAs (total of nine
RNA samples comprising three biological replicates) was hybridized to
the three available Affymetrix rat GeneChips (U34A, U34B, U34C;
experiment 1 in Fig. 1). 5,732 probe sets displayed statistically
significant differences (p < 0.05) between TGR and HO
cells and/or between HO and HOmyc3 cells. Adopting an
expression differential cutoff of
2-fold between the means of TGR and
HO and/or HO and HOmyc3 reduces the number of probe sets to 1,527. These probe sets were then grouped into four categories according to
their patterns of expression: 599 probe sets (39%) were categorized as
activated by Myc, 695 probe sets (46%) as repressed by Myc, 94 (6%)
as activated by overexpression of Myc, and 87 (6%) as repressed by
overexpression of Myc. Representative examples of each functional
category are shown in Fig. 4. The
remaining 52 probe sets (3%) exhibited patterns of expression whose
biological relevance to c-myc is not clear. To ascertain the
accuracy of the microarray analysis, we examined the mRNA
expression levels of 7 Myc-activated, 6 Myc-repressed, and 4 unaffected
genes using qPCR. In 17 of 17 cases the qPCR data confirmed the
microarray results. Because the U34A GeneChip contains most of the
known rat genes it was used in subsequent experiments.
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Fig. 4.
Categories of Myc target genes.
A, Myc-activated target. Expression is reduced in
c-myc /
(HO) cells relative to both
c-myc+/+ (TGR) and Myc-overexpressing (HOmyc3)
cells. U75392 corresponds to the prohibitin-2 gene. B,
Myc-repressed target. Expression is increased in
c-myc
/
(HO) cells relative to both
c-myc+/+ (TGR) and Myc-overexpressing (HOmyc3)
cells. S81478 corresponds to an oxidative stress-inducible
protein-tyrosine phosphatase gene. C, target activated by
Myc overexpression. Expression is increased in Myc-overexpressing
(HOmyc3) cells but is approximately equivalent in
c-myc+/+ (TGR) and
c-myc
/
(HO) cells. D10262 corresponds to the
choline kinase gene. D, target repressed by Myc
overexpression. Expression is decreased in Myc-overexpressing (HOmyc3)
cells but is approximately equivalent in
c-myc+/+ (TGR) and
c-myc
/
(HO) cells. Z78279 corresponds to the
type I procollagen
1 gene.
Genes responsive to Myc expression
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Fig. 5.
Patterns of MycER responsiveness in the
absence and presence of cycloheximide. A,
Myc-activated, early responding, direct target. Expression is inducible
with OHT and is statistically significant at the 4 h (and
subsequent) time points (upper panel). Expression of this
gene is affected by cycloheximide alone, but the effect is small
(~2-fold), and activation by OHT plus cycloheximide is both
statistically significant and 1.5-fold compared with the
cycloheximide only treatment (lower panel). AI178135 is
complement component 1, q subcomponent-binding protein. B,
Myc-activated, mid responding, indirect target. Expression is inducible
with OHT but is not statistically significant at the 2 h and
4 h time points (upper panel). Expression is not
affected by cycloheximide alone, and the difference between OHT plus
cycloheximide and cycloheximide only treatments is not statistically
different at any time point (lower panel). U60882 is an
arginine N-methyltransferase. C, Myc-repressed,
early responding target, showing a strong cycloheximide effect.
Expression is repressible with OHT and is statistically significant at
the 4 h and later time points (upper panel). Expression
is strongly affected by cycloheximide alone (almost 10-fold at 4 and
8 h time points). The difference between OHT plus cycloheximide
and cycloheximide only treatments is not statistically different at any
time point (lower panel). D15069 is adrenomedullin.
D, Myc-repressed, early responding target, showing a weak
cycloheximide effect. Expression is repressible with OHT and is
statistically significant at the 4 h and later time points
(upper panel). Expression is weakly affected by
cycloheximide alone (~2-fold at the 4 and 8 h time points). The
difference between OHT plus cycloheximide and cycloheximide only
treatments is not statistically different at any time point
(lower panel). This case illustrates the limitations caused
by the combined effects of cycloheximide treatment and the short
half-life of MycER. It could be argued that the repression caused by
OHT (~2-fold) should be discernible on top of a cycloheximide effect
of approximately the same magnitude and that this gene should thus be
classified as an indirect target. However, repression at 4 h is
relatively weak, whereas the cycloheximide effect is at its strongest.
Repression improves at the 8 h time point, but at this time
virtually no MycER is present in the cells. U02553 is a nonreceptor
type 16 protein-tyrosine phosphatase.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
(HO) cells relative to
c-myc+/+ (TGR) cells are reverted to a
c-myc+/+ pattern of expression in the HOmyc3
cell line.
/
(HO) cells, but only one was
OHT-responsive in MycER cells, indicating that they are likely to be
indirect targets. However, because many were weakly inducible at late
times in MycER104 cells, it is possible that ribosomal protein genes
can also respond to very high Myc levels, such as those found in tumor
cells (20).
B crystallin, identified as Myc-repressed in our analysis, have been
shown to function as negative effectors of apoptosis through their
ability to sequester cytochrome c from Apaf-1 (41) and
inhibit the maturation of caspase-3 (42), respectively. We also
identified a component of the mitochondrial permeability transition
pore complex as a Myc-repressed gene.
/
cells (43, 44).
-fodrin, the secretory carrier membrane protein-1, the
small G protein ARF2, and phospholipase D. In addition, proteins
involved in vesicle docking and fusion, including cellubrevin, Rab10,
the Rab effector GM130, and the Rab GDP-dissociation inhibitor, were
found to be repressed by Myc. In a general sense, a significant
fraction of Myc-repressed genes are involved in the interaction and
communication of cells with their external environment. It is
especially interesting to note that several of these targets have been
shown to possess tumor suppressor and antimetastatic properties. By
repressing genes involved in vesicular trafficking and cellular
adhesion inappropriate Myc expression may thus create a permissive
environment for aggressive tumor cell invasion.
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ACKNOWLEDGEMENTS |
---|
We thank Amy Whiting for help with computing analysis. We thank Dan Fraenkel and George Prendergast for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by United States Public Health Service Grant R01 GM-41690 from the National Institutes of Health (to J. M. S.). The Affymetrix GeneChip facility was supported by National Institutes of Health Grant RR-15578 from the COBRE Program of the National Center for Research Resources.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains illustrations of Myc-activated and
Myc-repressed genes.
Supported in part by National Institutes of Health Predoctoral
Training Grant GM-07601.
§ Present address: Dept. of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.
¶ Present address: University of California, San Francisco, CA 94143.
Present address: Dept. of Molecular Biology, Princeton
University, Princeton, NJ 08544.
** To whom correspondence should be addressed: J. W. Wilson Laboratory, Rm. 223, 69 Brown St., Providence, RI 02912. Tel.: 401-863-7631; Fax: 401-863-9653; E-mail: john_sedivy@brown.edu.
Published, JBC Papers in Press, January 14, 2003, DOI 10.1074/jbc.M210462200
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ABBREVIATIONS |
---|
The abbreviations used are: MycER, c-Myc-estrogen receptor fusion protein; BrdUrd, bromodeoxyuridine; CAD, trifunctional enzyme carbamoyl phosphate synthetase, aspartate transcarbamylase, dihydroorotase; GAPDH, glyceraldehyde phosphate dehydrogenase; OHT, 4-hydroxytamoxifen; qPCR, quantitative real time reverse transcription PCR.
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REFERENCES |
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