The Adrenomedullin Gene Is a Target for Negative Regulation by the Myc Transcription Complex
Xueyan Wang,
Mette A. Peters,
Fransiscus E. Utama,
Yuzhen Wang and
Elizabeth J. Taparowsky
Department of Biological Sciences Purdue University West
Lafayette, Indiana 47907-1392
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ABSTRACT
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The Myc family of transcription factors
plays a central role in vertebrate growth and development although
relatively few genetic targets of the Myc transcription complex have
been identified. In this study, we used mRNA differential display to
investigate gene expression changes induced by the overexpression of
the MC29 v-Myc oncoprotein in C3H10T1/2 mouse fibroblasts. We
identified the transcript of the adrenomedullin gene
(AM) as an mRNA that is specifically down-regulated in
v-Myc overexpressing C3H10T1/2 cell lines as well as in a Rat 1a cell
line inducible for c-Myc. Nucleotide sequence analysis of the mouse
AM promoter reveals the presence of consensus CAAT and TATA
boxes as well as an initiator element (INR) with significant sequence
similarity to the INR responsible for Myc-mediated repression of the
adenovirus major late promoter (AdMLP). Reporter gene assays confirm
that the region of the AM promoter containing the INR is
the target of Myc-mediated repression. Exogenous application of AM
peptide to quiescent C3H10T1/2 cultures does not stimulate growth, and
constitutive expression of AM mRNA in C3H10T1/2 cells
correlates with a reduced potential of the cells to be cotransformed by
v-Myc and oncogenic Ras p21. Additional studies showing that
AM mRNA is underrepresented in C3H10T1/2 cell lines stably
transformed by Ras p21 or adenovirus E1A suggest that AM
gene expression is incompatible with deregulated growth in this cell
line. We propose a model in which the repression of AM gene
expression by Myc is important to the role of this oncoprotein as a
potentiator of cellular transformation in C3H10T1/2 and perhaps other
cell lines.
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INTRODUCTION
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The myc family of protooncogenes encodes nuclear
proteins possessing a basic helix-loop-helix/leucine zipper domain
(bHLH/LZ) that mediates heterodimer formation with the bHLH/LZ protein
Max (1, 2, 3, 4, 5, 6, 7, 8, 9). The Myc-Max protein complex binds to target DNA with a core
consensus of CACGTG (1, 3, 4, 5, 8) and stimulates transcription of
synthetic or natural promoters containing these sites (3, 10, 11).
Since Myc protein expression is a feature of proliferating cells
(12, 13, 14), Myc-Max DNA-binding complexes predominate in growing cells
and presumably this results in the elevated expression of Myc target
genes. Interestingly, Max levels are not regulated in response to
changes in cellular growth conditions (4, 15, 16, 17, 18, 19), and in
differentiating or quiescent cells, Max forms heterodimers with a
number of other bHLH/LZ proteins. These complexes possess the same
DNA-binding preference as Myc-Max heterodimers yet actively inhibit,
rather than activate, target gene transcription (20, 21, 22, 23).
The biochemical characterization of Myc, Max, and their related bHLH/LZ
family members has outpaced our ability to fully comprehend why
deregulated expression of Myc is a common feature of tumor tissue and
causes cells in culture to adopt characteristics that propel them
toward a phenotype of uncontrolled growth. The key to understanding Myc
protein function is to identify the full complement of genes whose
transcription is influenced by the overexpression of Myc. Toward that
goal, a number of strategies have been used and several genetic targets
for the Myc protein complex have been found (reviewed in Refs. 24, 25, 26).
As predicted by the observed transactivation of synthetic reporter
genes by Myc-Max heterodimers, the vast majority of Myc targets
identified to date are regulated positively by Myc and include
-prothymosin, ODC, cad,
p53, ECA39, gadd 45,
cdc25A, and LDH-A (reviewed in Refs. 24, 25, 26).
Transcription activation of these genes by Myc requires DNA binding and
thus relies on the integrity of the carboxyl terminal bHLH/LZ motif
that mediates binding to Max and binding to DNA (1, 8, 9, 27).
Transcription activation by Myc also requires sequences in the
amino-terminal transcription activation domain (TAD) of Myc (5, 7, 28)
but, curiously, these sequences do not overlap completely with the TAD
sequences important for cellular transformation by Myc. Experiments
performed by Cole and colleagues (29) demonstrated that a c-Myc protein
modified by point mutation in the highly conserved Myc box II region of
the TAD retained the ability to transactivate gene expression, yet did
not function in cellular transformation assays. In contrast, there are
only a few genes that have been found to be regulated negatively by Myc
(reviewed in Refs. 24, 25, 26). Repressed target genes include the
c-myc gene (30), thrombospondin-1 (31), and the
adenovirus major late promoter (AdMLP), which represents the most
studied model of transcriptional repression by Myc (32, 33). The
mechanism by which Myc negatively regulates the expression of genes is
controversial and, in some cases, does not appear to require
heterodimerization with Max (34). However, transcription repression by
Myc does depend on sequences in the amino-terminal TAD of Myc and, in
many cases, is abolished by the same point mutation in Myc box II that
adversely affects cellular transformation by Myc (30, 32, 33).
Recognizing the need to identify additional genetic targets of Myc that
may be important for cellular transformation, we used mRNA differential
display (35, 36) to evaluate gene expression changes triggered by Myc.
The starting material for the study was RNA isolated from C3H10T1/2
mouse fibroblasts (10T1/2) and from a C3H10T1/2 derivative, myc
neo 13A, which constitutively expresses the v-Myc oncoprotein.
Myc neo 13A cells were characterized previously as a line
harboring Myc-induced genetic changes that do not grossly alter cell
morphology or growth rate, but predispose the cells to transformation
by oncogenic Ras p21 (37). Comparison of several displays resulted in
the identification of a transcript from the adrenomedullin
gene (AM) that is present in 10T1/2 cells, but is totally
absent in myc neo 13A cells. The display results were
confirmed by Northern blot analysis of RNA isolated from these and
other v-Myc- and c-Myc-overexpressing cell lines. Reporter gene assays
demonstrate that the promoter of the mouse AM gene is the
target for Myc-mediated repression.
The first AM gene to be characterized was isolated from
human tissue in 1994 (38) and encodes two secreted proteins,
adrenomedullin (AM) and proadrenomedullin peptide (PAMP), both of which
function as vasodilators in the circulating plasma (39, 40). AM is a
member of the calcitonin family of peptides and signals through
G-protein coupled, seven-transmembrane receptors that increase
intracellular levels of cAMP (41, 42). Thus, like cAMP, AM exerts
positive and negative effects on the growth of normal cells and tumor
tissues (reviewed in Ref. 43). In this study, we show that there is an
inverse correlation between levels of AM expression and the
growth potential of C3H10T1/2 cells, and we demonstrate experimentally
that forced expression of AM mRNA inhibits cotransformation
by oncogenic Ras p21 and v-Myc. We have identified the AM
gene as a novel candidate for transcriptional regulation by Myc and the
AM promoter as a valuable tool for investigating the
molecular mechanism through which Myc represses cellular genes. Our
studies in C3H10T1/2 cells support a role for AM as an
inhibitor of cell growth and suggest that the peptides encoded by this
gene could be used in autocrine, paracrine, or endocrine strategies to
control the proliferation of specific cell types.
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RESULTS
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Myc Down-Regulates AM mRNA Expression in C3H10T1/2 and
Rat 1a Fibroblasts
Stable overexpression of either the v-myc or the
c-myc oncogene in C3H10T1/2 (10T1/2) mouse fibroblasts does
not alter significantly the growth properties of the cells, but does
induce intracellular changes that increase the frequency of
transformation by the H-ras oncogene (44). The nature of the
changes resulting from the overexpression of Myc in mammalian cells is
not completely known. However, since Myc participates in a nuclear
protein complex that binds DNA and influences gene transcription, it is
likely that the activation and/or repression of a specific set of
cellular genes is critical for Myc to mediate its dramatic effects on
cell growth.
To identify genetic targets of transcriptional regulation by Myc, our
laboratory used the mRNA differential display technique (35, 36) to
detect mRNA molecules that are differentially expressed in 10T1/2 cells
and in myc neo 13A cells, a 10T1/2 cell derivative
constitutively expressing the MC29 v-Myc protein. Total RNA isolated
from both cell types was reverse transcribed using four distinct oligo
(dT) primers, and the resulting cDNA was amplified in the presence of
[35S]dATP using PCR with the same oligo dT primers and a
random 10-mer primer (see Materials and Methods for
details). After resolution on a denaturing polyacrylamide gel, the
radiolabeled DNA fragments generated by the PCR were compared. One
fragment, designated MRG4 for Myc-responsive gene 4, was amplified in
several independent samples prepared from wild-type 10T1/2 cells, but
was never detected in samples obtained from myc neo 13A
cells (Fig. 1A
and additional data not
shown). MRG4 was purified from the dried gel and reamplified. Northern
blot analysis of polyA+ mRNA isolated from 10T1/2 and
myc neo 13 A cells using a [32P]dCTP-labeled
MRG4 probe revealed that this cDNA recognized a 1.6-kb mRNA that is
expressed in 10T1/2 cells and is absent in myc neo 13A cells
(Fig. 1B
).

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Figure 1. Differential Expression of mRNA in C3HIOT1/2
vs. myc nec 13A Cells
A, Differential display detecting MRG4 as a cDNA fragment representing
mRNA differentially expressed in C3H10T1/2 (lanes 15)
vs. myc neo 13 A cells (lanes 610).
Total RNA isolated from both cellular sources was reverse transcribed,
and the resulting first-strand cDNA was amplified by PCR in separate
reactions as described in Materials and Methods. The
MRG4 band was detected in three of five 10T1/2 samples but not in any
of the myc neo 13 A samples. B, The MRG4 cDNA fragment
was excised from the gel, reamplified, and used as a Northern blot
hybridization probe as described in Materials and
Methods. The MRG4 probe detected a 1.6-kb mRNA species
expressed in 10T1/2 cells, but not in myc neo 13 A cells
(upper panel). The migration of the 18S and 28S rRNA was
determined by EtBr staining of the gel before transfer and is indicated
on the right. The membrane was rehybridized with a probe
for G3PDH mRNA to control for RNA loading (lower
panel).
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To establish the identity of the transcript represented by MRG4, the
100-bp cDNA fragment was cloned and sequenced. A search of the database
revealed that the mouse MRG4 sequence shares greater than 95% identity
with the 3'-untranslated region (UTR) of the rat AM cDNA
(45). The full-length rat AM cDNA was used as a
32P-dCTP-labeled probe for Northern blot analysis of 10T1/2
and myc neo 13A mRNA. As shown in Fig. 2A
, the rat AM cDNA detects a
similarly sized mRNA as the MRG4 probe. The mouse AM mRNA is
expressed in wild-type 10T1/2 cells and is not detected in the RNA
prepped from myc neo 13A cells. This observation was not
unique to the myc neo 13 A cell line. mRNA was isolated from
a second 10T1/2 v-myc cell line (myc neo 10) and
from colonies pooled after the stable transfection of 10T1/2 cells with
a neomycin resistance gene (neo pool) or with the neomycin
resistance gene and MC29 v-myc (v-myc pool) (Fig. 2B
). Northern analysis of these additional RNA samples with the
AM cDNA probe confirmed that AM mRNA is decreased
significantly in all of the v-Myc-expressing cell lines. RT-PCR was
used to estimate the levels of v-myc mRNA in the myc
neo 13A, myc neo 10, and v-myc pool samples
(Fig. 2C
), and the results reveal an inverse relationship between the
level of v-myc mRNA expressed and the level of mouse
AM mRNA detected in each sample. RNA also was prepared from
Rat 1a cells that constitutively express either a human c-Myc-estrogen
receptor (Myc-ER) fusion protein or the estrogen receptor (ER) alone
(control) (Fig. 3
). Growth of these cells
in the presence of 4-hydroxytamoxifen (TM) results in the translocation
of the ER and the Myc-ER from the cytosol to the nucleus. Northern
analysis using the rat AM probe on RNA isolated from ER and
Myc-ER cells, treated or untreated with TM, established that
AM mRNA expression decreases after nuclear translocation of
the Myc-ER and the concomitant induction of c-Myc activity in cells
(Fig. 3
). Therefore, we conclude that the overexpression of Myc in
C3H10T1/2 and Rat 1a cells leads to the down-regulation of
AM mRNA expression.

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Figure 2. Northern Blot Hybridization Demonstrating that
AM mRNA is Underrepresented in 10T1/2 cells Expressing
v-Myc
A, Poly A+ mRNA from 10T1/2 and myc neo 13 A
cells was hybridized with the rat AM cDNA probe
(upper panel), stripped, and rehybridized with a probe
for G3PDH mRNA to control for RNA loading (lower
panel). The migration of the 18S and 28S rRNA was determined by
EtBr staining of the gel before transfer and is indicated on the
right. B, Total RNA isolated from 10T1/2 cells, the
myc neo 10 cell line, and pools of 500 G418-resistant
clones representing 10T1/2 cells stably transfected with
pKOneo DNA alone (neo pool) or
pKOneo plus the v-myc gene
(v-myc pool) was hybridized sequentially with rat
AM (upper panel) and G3PDH
(lower panel) probes as described in panel A. C, RT-PCR
was performed on total RNA from the cellular sources described in
panels A and B using oligonucleotide primers specific for
v-myc (upper panel) and for ß-actin
(lower panel). Lane M contains DNA size markers from a
HindIII digest of bacteriophage DNA. Results suggest
an inverse correlation between the amount of v-myc mRNA
expressed and the extent to which AM mRNA levels are
repressed.
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Figure 3. Northern Blot Analysis of AM mRNA
Expression in Rat 1a Cells Containing an Inducible c-Myc Protein
(Myc-ER)
Total RNA was isolated from proliferating control cells (ER) and Myc-ER
cells that had been mock treated with EtOH (-) or treated with TM (+)
as described in Materials and Methods to induce nuclear
translocation of the ER or Myc-ER proteins. Northern blot hybridization
was performed as in Fig. 1B using rat AM (upper
panel) and G3PDH (lower panel)
probes. The migration of the 18S rRNA was determined by EtBr staining
of the gel before transfer and is indicated on the left of the
upper panel.
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The AM Promoter Is the Target of
v-Myc-Mediated Gene Repression
Multiple molecular mechanisms could underlie the
observed correlation between the overexpression of Myc and the
down-regulation of endogenous AM mRNA. To establish whether
repression occurs by altering the transcriptional activity of the
AM gene, we isolated a 6.5-kb genomic DNA fragment that
contains the mouse AM gene. Our own DNA sequence analysis,
supplemented with published information available on the mouse
AM gene (46), revealed that the mouse AM gene has
a structure similar to that of the human AM gene (38) and
that the coding sequences for mouse, rat, pig, and human AM
genes are strikingly similar (38, 45, 46, 47, 48). As a first step toward
examining transcriptional regulation of the mouse AM gene, a
3.0-kb DNA fragment mapping 5' to the methionine initiator codon was
inserted into the pGL2 basic plasmid directly upstream of
luciferase-coding sequences to generate AM-Luc. 10T1/2 cells were
transiently transfected with either AM-Luc, pGL2 basic, or pGL2 basic
driven by the SV40 promoter and, after 48 h, cell extracts were
prepared and assayed for luciferase activity. Results revealed that the
AM 5'-flanking region drives luciferase expression as well
as the SV40 promoter (data not shown) and thus contains the
cis-acting DNA elements necessary for directing
AM gene transcription in C3H10T1/2 cells. 10T1/2 cells then
were transfected with AM-Luc and increasing amounts of pMC29, an
expression vector for v-Myc (Fig. 4A
).
The observed dose-dependent effect of v-Myc on AM-Luc expression
indicates that the 3.0-kb AM promoter fragment also contains
the cis-acting DNA elements that are responsible for
Myc-mediated transcriptional repression.

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Figure 4. Reporter Genes Controlled Transcriptionally by the
Mouse AM Promoter Are Repressed by Wild-Type v-Myc, but
Not by B-ATF or a Nonfunctional v-Myc Protein
A, 10T1/2 cells were transiently cotransfected with 1 µg of
AM-Luc, a luciferase reporter gene regulated by 3 kb of 5'-flanking
sequence from the mouse AM gene, and the indicated
amount (in micrograms) of pMC29, a v-myc expression
plasmid. Luciferase activities in each cellular extract were normalized
to total protein content and are expressed as a percentage of the
activity in control cells transfected with AM-Luc alone. Each value
represents the average of at least four independent determinations, and
the error bars indicate the SEM. B, 10T1/2
cells were transiently cotransfected with 2.5 µg of AM-CAT, a CAT
reporter gene regulated by 2 kb of 5'-flanking sequence from the
mouse AM gene, and 1.25, 2.5, or 7.5 µg of an
expression plasmid for either v-Myc, B-ATF, or a nonfunctional v-Myc
protein ( Myc). The CAT activity present in each extract was
normalized using total protein content and is expressed as a percentage
of the activity in control cells transfected with AM-CAT alone. Each
value represents the average of at least four independent
determinations, and the error bars indicate the
SEM.
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To analyze the specificity of AM promoter regulation by Myc,
a second construct containing a chloramphenicol acetyl transferase
(CAT) reporter gene under the transcriptional control of a 2.0-kb
fragment mapping 5' to the mouse AM methionine initiator
codon was constructed (AM-CAT). This reporter gene was active in 10T1/2
cells and responded in a manner similar to AM-Luc when cointroduced
with increasing amounts of v-Myc (Fig. 4B
). Additional transfections
demonstrated that coexpression of a nuclear localized v-Myc protein
deleted of all but the first 84 amino-terminal residues of Myc
(
Myc), or of a transcription factor unrelated to Myc (B-ATF), does
not cause a significant drop in AM-CAT activity, indicating that the
repression noted with wild-type v-Myc is specific (Fig. 4B
).
The region of the mouse AM promoter that is responsive to
Myc is presented in Fig. 5
along with the
overall exon-intron structure of the mouse AM gene. The
promoter contains consensus CAAT and TATA boxes and a 10-bp sequence
positioned 20 bp 3' to the TATA box that fits the consensus nucleotide
sequence of an initiator region, or INR (49). Interestingly, INR
elements are found in other genes that are repressed transcriptionally
by Myc (32, 50). The transcription start point of the mouse
AM promoter has been mapped by Okazaki and colleagues (46)
to an A residue immediately 3' to the INR sequence (46), confirming its
role as the initiator region of the mouse AM gene.

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Figure 5. Organization of the 6.5-kb EcoRI
Genomic DNA Fragment Spanning the Mouse AM Gene
Solid squares represent exons; ATG indicates the point
of translational initiation 23 bp into exon 2. Below is
the nucleotide sequence of the AM promoter region with
the CAAT box, the TATA box, and potential INR (CTCACTAGTC)
underlined. The bold arrow indicates the
point of transcriptional initiation (46 ). Diagrammed
above the AM gene are the AM-Luc and
AM-CAT reporter constructs, which are under the transcriptional control
of an AM 5'-flanking region extending from upstream of
the ATG to the EcoRI or BglII restriction
enzyme site, respectively. Bent arrows represent the
sequence information contained within the 0.73-CAT, 0.62-CAT, and
(-INR)-CAT reporters used in Fig. 6 .
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Additional deletions were generated to examine the role of the
INR in AM promoter function (Fig. 5
). 0.73-CAT contains 110
bp 5' to the start site of the gene and thus contains the INR, TATA,
and CAAT motifs. 0.62-CAT is deleted for all of these elements,
including 8 of the 10 bp comprising the INR. (-INR)-CAT)
contains the DNA sequence 5' to the central C nucleotide within the
INR, terminating at the same upstream site as 0.73-CAT. These
constructs were tested for transcriptional activity in 10T1/2 cells
using the AM-CAT construct as the positive control (Fig. 6A
). 0.73-CAT expresses at a level
slightly above AM-CAT and, as predicted, the severely deleted 0.62-CAT
construct is essentially inactive in these assays. (-INR)-CAT is
expressed at a low, intermediate level, generating approximately 25%
of the CAT activity of the positive control. Interestingly, when v-Myc
is coexpressed with these reporters, 0.73-CAT is repressed more than
70%, while (-INR)-CAT is repressed only 30% (Fig. 6B
). We believe
these results provide evidence that the INR consensus element plays an
important role in the overall activity of the AM promoter as
well as in the targeted repression of AM gene expression by
Myc.

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Figure 6. Efficient Expression and Myc-Mediated Transcription
Repression of the AM Gene Maps to a Region of the
Promoter Containing the INR
10T1/2 cells were transiently transfected with 5 µg of the indicated
reporter gene and, for panel B, 5 µg of vector DNA (-Myc) or 5 µg
of pMC29 (+ Myc). CAT activity in each extract was normalized using
total protein content and is expressed as a percentage of the activity
in control cells. In panel A, the activity from AM-CAT was set at
100%. For panel B, the activity of each reporter gene in the absence
of Myc was set at 100%, even though (-INR)-CAT directs only 25% of
the activity of 0.73-CAT (panel A). Values presented are the average of
results obtained from at least three independent determinations.
Error bars indicate the SEM.
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AM Is Not Mitogenic for C3H10T1/2 Fibroblasts and Blocks Focus
Formation by Oncogenic H-Ras and v-Myc
The observation that Myc overexpression correlates with
transcriptional repression of the AM gene suggests that AM
expression may be incompatible with enhanced proliferation. In this
regard, however, it has been shown that AM is mitogenic for several
cell types (reviewed in Ref. 43), including Swiss 3T3 fibroblasts (51).
To examine the influence of AM on 10T1/2 cell proliferation, we treated
quiescent 10T1/2 fibroblasts or Swiss 3T3 cells with 100 nM
AM peptide as described in Materials and Methods.
Proliferation was measured by BrdU uptake and quantitated after
immunostaining of fixed cultures with a BrdU antibody. As shown in Fig. 7A
, the Swiss 3T3 cells responded as
expected with AM stimulating growth to 50% of the level obtained with
10% FBS (set at 100%). In contrast, the 10T1/2 cells were more
difficult to render quiescent and after exposure to AM displayed even
lower levels of BrdU incorporation than the 0% serum-treated control
(Fig. 7B
). Since 10T1/2 cells express AM receptors (F. Cuttitta,
personal communication), it appears that this cell line joins a number
of others in which AM-induced signaling is not correlated with cell
growth (52, 53, 54, 55).

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Figure 7. AM Stimulates the Growth of Swiss 3T3 Fibroblasts,
but Not 10T1/2 Cells
A, Quiescent Swiss 3T3 cultures were incubated with the indicated
media, and proliferation was measured by BrdU incorporation as
described in Materials and Methods. The percent of
BrdU-positive cells is expressed relative to the control group (100%),
which was stimulated with 10% bovine calf serum. B, Quiescent
C3H10T1/2 cultures were incubated with the indicated media and
proliferation was measured as in panel A. The percent of BrdU-positive
cells is expressed relative to the control group (100%), which was
stimulated with 10% FBS. The composition of the medium used to treat
each cell type, along with the final concentrations of adrenomedullin
peptide (AM), IBMX, and insulin (INS), is described in Materials
and Methods. Each experiment was repeated three times, with a
total of 10 independent fields scored per group per experiment.
Error bars represent the SEM.
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The apparent role of AM as an agonist to 10T1/2 cell growth was tested
further in transformation assays. The C3H10T1/2 cell line is an
established model system in which the transforming effects of the human
H-ras oncogene are enhanced 2- to 3-fold by coexpressing a
collaborating oncogene, such as v-myc, which displays no
potential to morphologically transform 10T1/2 cells on its own (7, 44, 56). C3H10T1/2 cells were stably transfected as described in
Materials and Methods with expression vectors for oncogenic
Ras p21, v-Myc, and rat AM. The cells were cultured for approximately 2
weeks in reduced serum medium to select for transformed foci (Table 1
). The expression of oncogenic Ras and
v-Myc results in a level of focus formation that is set at 100%.
Transfection of cells with Ras, v-Myc, and either 1.8 µg or 6.0 µg
of DNA-encoding rat AM reduces the effect of v-Myc on focus formation
by 32% and 54%, respectively. These results are consistent with the
results from the proliferation assays and suggest that one effect of
Myc as a collaborating oncoprotein may be to reduce the negative impact
of AM on the transformation process.
AM mRNA Is Underrepresented in C3H10T1/2 Cell Lines
Transformed by the Expression of Oncogenic H-Ras or Adenovirus E1A
To extend the apparent inverse correlation between AMgene expression and enhanced 10T1/2 cell growth, we examined the
expression pattern of mouse AM mRNA in several other cell
lines previously characterized by our laboratory. Ras neo
11A is a 10T1/2 derivative selected on the basis of morphological
transformation and certain biochemical parameters associated with the
expression of high levels of mutationally activated H-Ras p21 (57, 58).
Similarly, EN-2 is a morphologically transformed, 10T1/2 cell line that
expresses the human adenovirus E1A 12S and 13S proteins and grows
extremely well in anchorage-independent growth assays (37). B-ATF-4 is
a 10T1/2 cell line that overexpresses B-ATF, a basic leucine zipper
transcription factor that is a potent dominant-negative to AP-1 and a
potential tumor suppressor (Ref. 59 and D. Echlin and E. Taparowsky, in
preparation). PolyA+ mRNA was isolated from these cell
lines and analyzed along with RNA prepared from wild-type 10T1/2 and
myc neo 13A cells for the expression of mouse AM
mRNA. As shown in Fig. 8
, AM
gene expression is extinguished in the cell lines carrying an oncogene,
while the line expressing B-ATF shows a higher level of AM
mRNA. In the case of the ras neo 11A cells, the absence of
AM mRNA appears to be a consequence (or prerequisite) of
selecting a stable, Ras-transformed phenotype, since we do not detect
reduced CAT activity in extracts obtained from 10T1/2 cells
cotransfected with AM-CAT and pT24, an expression vector for oncogenic
H-Ras p21 (data not shown). It remains to be determined whether
alterations in endogenous c-Myc expression are linked to the lack of
AM mRNA in the ras neo 11A and/or EN-2 cell
lines. However, regardless of the regulatory mechanism, these
observations provide additional evidence of an inverse relationship
between the proliferative potential of 10T1/2 cells and AM
expression and suggest that the negative regulation of AM
transcription by Myc might be a key event facilitating cellular
transformation by other oncoproteins.

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Figure 8. Mouse AM mRNA Is Underrepresented in
10T1/2 Cells Expressing v-Myc, Activated H-Ras p21, or the Adenovirus
E1A Oncoprotein
Total RNA was isolated from 10T1/2 cells stably coexpressing a
selectable marker gene and either v-Myc (myc neo 13A),
H-Ras p21 (ras neo 11A), E1A (EN-2), or B-ATF (B-ATF).
Northern blot hybridization with the AM cDNA probe was
performed as described in Fig. 1B (upper panel). A
photograph of the EtBr-stained gel before blotting (lower
panel) shows the positions of the 18S and 28S rRNA, which are
indicated to the right of each panel.
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DISCUSSION
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For several years, it has been recognized that the Myc proteins
participate in nuclear DNA-binding complexes that regulate gene
transcription. Significant effort has been devoted to identifying the
cellular target genes whose expression is influenced by Myc with the
dual purpose of deciphering the molecular basis of Myc action and of
understanding why deregulated Myc expression has such dramatic
consequences on vertebrate growth and development. The vast majority of
the genetic targets identified for Myc are regulated positively in the
presence of the protein, and most encode proteins that are associated
with cellular growth. These genes include ODC,
p53, cad, LDH-A, cyclin A, cyclin E,
and cdc25 A, and for some of the most intriguing, such as
the cyclins, it remains unclear whether the genes are direct targets of
Myc or are up-regulated as a secondary consequence of Myc activity in
specialized cell types (reviewed in Refs. 24, 25, 26). Far fewer genetic
targets have been described that are regulated negatively in the
presence of Myc (reviewed in Refs. 24, 25, 26) and, in fact, the best
studied models of Myc repression are the phenomena of c-myc
autoregulation (30) and repression of the AdMLP (32, 33). The need to
identify targets of Myc-mediated transcription repression is
particularly important, since structure/function studies of Myc reveal
that the amino-terminal sequences essential for cellular transformation
by Myc colocalize with the motif most critical to transcription
repression by Myc (29, 30, 32). Thus, to fully understand the
physiological consequences of Myc overexpression in tumor development,
the products of the genes that are down-regulated by Myc hold the most
promise.
In this study, we have identified the AM gene as a genetic
target for negative regulation by Myc. AM mRNA is reduced in
C3H10T1/2 cell lines overexpressing v-Myc and in Rat 1a cells in which
c-Myc function is conditional upon treatment with TM. Using the AM-CAT
reporter gene, which is expressed very well in 10T1/2 cells, we show
that coexpression of wild-type v-Myc efficiently represses
AM promoter activity. The mouse AM promoter
contains consensus CAAT and TATA boxes and a potential INR (CTCACTAGTC)
with a high degree of sequence similarity to the INR of the AdMLP
(CTCACTCTCT) (32). Using additional truncations of the AM
promoter, it is clear that the INR is necessary for full promoter
activity since a construct lacking the INR drives CAT expression to a
mere 25% of control levels. However, this remaining 25% activity is
not repressed significantly by Myc (Fig. 6B
), suggesting that the INR
also is the major target of Myc-mediated repression of this promoter.
Although our studies do not resolve whether the effect of Myc on
AM mRNA expression is direct or indirect, the similarity of
the AM promoter to the AdMLP suggests that the same
mechanism of regulation will be involved (32). Thus, future studies
will address whether heterodimer formation through the Myc HLH/LZ motif
is required for this repression or whether overexpression of the
bHLH/LZ protein USF counteracts the negative influence of Myc on
AM gene expression.
In addition to identifying the mouse AM promoter as a DNA
element that can be exploited to study the molecular mechanism of
Myc-mediated gene repression, our goal in undertaking this study was to
identify genetic targets of Myc whose protein products may play a role
in cellular transformation by Myc. The AM gene encodes a
preproadrenomedullin precursor protein that is processed to give rise
to two biologically active proteins, the 50-amino acid AM peptide and a
structurally unrelated, 20-amino acid PAMP N-terminal peptide (38, 39, 40, 45, 60). The AM peptide was isolated originally from cell extracts of
human pheochromocytoma tissue using its ability to induce cAMP
production in platelets as an assay (39). Both AM and PAMP function as
hypotensive peptides (39, 40), and AM exerts profound effects on
hormone production by certain tissues (61, 62). A concerted effort has
been made to understand the role of AM production and AM
receptor-mediated signaling in normal cells and in cancer cells. To
date, although much of the available data support a role for AM as an
autostimulator of cell growth, some studies demonstrate that AM
inhibits mitogenesis in rat mesangial cells and vascular smooth muscle
cells (52, 53, 54) and that both AM and PAMP can inhibit the growth of
neuroblastoma cells (63). The tissue-specific effects of AM can be
explained, in part, by the fact that AM signals through G-protein
coupled, transmembrane receptor proteins that trigger intracellular
cAMP production (41, 42). It is well known that cAMP activation of
protein kinase A (PKA) exerts a variety of biological effects depending
on the cellular background. For example, while PKA stimulates
mitogenesis in the pituitary and the expression of the c-fos
gene in PC12 cells (reviewed in Ref. 64), PKA modifies the Raf-1 kinase
in fibroblasts and antagonizes mitogenesis induced by activated Ras p21
(65).
The mRNAs for preproadrenomedullin (Fig. 2
) and for the two major
species of AM receptor (F. Cuttitta, personal communication) are
expressed in 10T1/2 cells, although experiments to confirm protein
production have not been performed. Exogenous application of AM to
serum-starved 10T1/2 cells does not drive cell cycle entry as it does
for Swiss 3T3 cells and may even enhance 10T1/2 growth arrest (Fig. 7B
). In studies not presented here, we have used colony assays to test
whether AM overexpression is toxic to 10T1/2 cells and have
observed no adverse effect of AM on long-term cell viability. However,
AM overexpression does reduce the frequency of cellular
cotransformation by Myc and Ras by an average of 50% (Table 1
), and AM gene expression is
extinguished in 10T1/2 cell lines selected over time on the basis of
their transformed growth properties (Fig. 8
). These observations prompt
us to speculate that the autocrine stimulation of PKA activity by AM
production in 10T1/2 cells antagonizes the signaling pathways that
normally promote unrestricted cell growth in this cell type. Additional
experiments to measure AM receptor-ligand interaction in 10T1/2 cells
and to characterize the intracellular second messenger molecules
activated as a result of this interaction will address further the
validity of this hypothesis.
While genetic targets for Myc activity continue to be identified,
several trends are emerging. Perhaps the most obvious is the wide
variety of target genes identified to be either positively or
negatively regulated by Myc, with the products of these genes playing
diverse roles in cellular growth and metabolism. While one
interpretation of these data would be that the "true" Myc target is
yet to be found, a second interpretation is that Myc overexpression
exerts pleiotropic effects in cells, and that the precise combination
of gene expression changes required to alter cellular growth is
different for each cell type. This idea is supported by a recent study
demonstrating that the most logical candidate for a universal target of
positive regulation by the Myc/Max transcription complex, the
cdc25 A gene, is not induced under all conditions where Myc
expression is directly responsible for deregulating cell growth (66).
It is apparent that additional studies will be required before a
complete understanding of how Myc functions to control cell growth and
induce cellular transformation is obtained.
 |
MATERIALS AND METHODS
|
---|
Cell Lines
C3H10T1/2 mouse embryo fibroblasts (10T1/2) (ATCC CCL226)
(American Type Culture Collection, Manassas, VA) were maintained in
basal modified Eagles medium (GIBCO, Grand Island, NY)
supplemented with 10% FBS (Bio-Whittaker, Walkersville, MA), 100 U/ml
penicillin, and 100 µg/ml streptomycin (GIBCO). Swiss 3T3 fibroblasts
(ATCC CCL92) were maintained in high-glucose DMEM (GIBCO) supplemented
with 10% bovine calf serum (Hyclone Laboratories, Logan, UT), 100 U/ml
penicillin, and 100 µg/ml streptomycin (GIBCO). The myc
neo 13A, ras neo 11A, and EN-2 cell lines have been
described previously (44, 57, 37) and were maintained as described for
10T1/2, except in complete medium supplemented with 400 µg/ml G418
(GIBCO). B-ATF-4, a 10T1/2 cell line overexpressing the basic leucine
zipper transcription factor B-ATF, was generated after the stable
transfection of 10T1/2 cells with pDCR-B-ATF (59) and the selection of
neomycin-resistant colonies as described previously (56). Stable
transfection of 10T1/2 cells also was used to establish the pooled
populations (
500 individual clones) of neomycin-resistant cells or
of neomycin-resistant/v-Myc-expressing cells used in this study. The
Rat 1a Myc-ER cell line (obtained from Dr. Stephen R. Hann, Vanderbilt
University, Nashville, TN) has been described previously (67) and was
maintained in high-glucose DMEM supplemented with 10% FBS, 100 U/ml
penicillin, 100 µg/ml streptomycin, and 400 µg/ml G418. To measure
the effects of induced Myc activity in the Rat 1a Myc-ER cells,
subconfluent cultures were treated for 20 h with 500 ng/ml TM
(Research Biochemicals, Inc., Natick, MA) dissolved in ethanol.
Control cultures were treated for an equivalent length of time with
ethanol alone.
Plasmids
A P1 genomic clone containing the mouse AM gene was
isolated by Genome Systems (St. Louis, MO) using a fragment of the
mouse AM cDNA as a probe. A 6.5-kb EcoRI fragment
containing the AM-coding region plus 3.0 kb of 5'- and 2.6 kb of
3'-flanking sequence was identified by Southern blot hybridization and
cloned into Bluescript KS+ (Stratagene, La Jolla, CA) to
generate pBSKSmgAM/R13. The exon-intron structure of the mouse
AM gene was determined by dideoxy nucleotide chain
termination sequencing (Sequenase 2.0, Amersham, Cleveland, OH)
in the presence of [35S]dATP (1000 Ci/mmol, Amersham).
The complete genomic DNA sequence of the mouse AM gene was
published previously (46). A PCR-generated, BamHI-tailed,
DNA fragment spanning 3.0 kb of sequence 5' to the mouse AM
ATG codon was cloned into the BglII site of pGL2 basic
(Promega, Madison, WI) to create pGL2BmgAM5'-3 (AM-Luc). A 2.0-kb
BglII/BamHI fragment deleting 1.0 kb from the
5'-end of the 3.0-kb BamHI fragment in AM-Luc was
blunt-ended and used to replace the E1B promoter in E1B TATA CAT (68)
to generate mgAM5'-2 kbCAT (AM-CAT). AM-CAT was restricted with
XhoI and PstI, blunt-ended, and religated to
generate 0.73-CAT. A SpeI restriction site internal to the
consensus INR in the promoter (Fig. 5
) was used to construct 0.62-CAT
and (-INR)-CAT. The pMC29, pT24, and pKOneo plasmids have
been described previously (56), as have pDCR-B-ATF (59) and
84/NLS
(
Myc) (69). PDCR-rAM, which contains the coding region of rat
AM under the control of the cytomegalovirus promoter,
was generated by PCR from pcDNA-rAM, which was obtained from Dr. J.
Sakata (Miyazaki Medical School, Miazaki, Japan).
mRNA Differential Display
Total RNA was isolated from proliferating 10T1/2 and myc
neo 13A cells as described (70, 44), treated with
deoxyribonuclease I (DNase I) (0.2 U/µl) for 30 min at 37 C and,
after inactivation of the DNase by heating to 95 C for 5 min, used for
mRNA differential display (35, 36) (GenHunter Corp., Brookline, MA).
Briefly, 200 ng of RNA were reverse transcribed into first-strand cDNA
using four different 3'-primers provided by GenHunter (T12MG, T12MA,
T12MT, and T12MC). Replicate RT reactions were performed on each RNA
sample to obtain duplicate cDNA pools. The pools were amplified by PCR
using the same T12M 3'-primer and a synthetic 5'-primer (AP-2) designed
and provided by GenHunter. The PCR was carried out for 40 cycles at 94
C for 30 sec, 40 C for 2 min, and 72 C for 30 sec in a total volume of
20 µl containing 1.0 µl of [35S]dATP (1000 Ci/mmol;
Amersham), 0.2 µl of AmpliTaq DNA polymerase (Perkin-Elmer,
Branchburg, NJ), and deoxynucleoside triphosphates (dNTPs) at a
concentration of 2.0 µM. The radiolabeled PCR products
were resolved by electrophoresis through a 6% denaturing
polyacrylamide gel and visualized by autoradiography. The MRG4 fragment
was excised from the gel, eluted by soaking in 10 mM Tris,
1 mM EDTA, pH 7.4, concentrated by ethanol precipitation,
and reamplified by PCR using the primers and conditions described
above, except that the dNTP concentration was adjusted to 20
µM. Amplified MRG4 DNA was used as a hybridization probe
for Northern blots (described below) and cloned into Bluescript
KS+ using EcoRI and BamHI linkers to
generate pBS-MRG412. The nucleotide sequence of MRG4 was determined
using [35S]dATP (1000 Ci/mmol, Amersham) and the
Sequenase 2.0 Kit (Amersham).
Northern Blot Hybridization
A hybridization probe representing the coding region of the rat
AM cDNA was obtained by restricting pDCR-rAM with
SalI and BamHI and isolating the 555-bp insert
fragment. Total RNA was isolated from the indicated sources as
described previously (70, 44), and poly A+ mRNA was
isolated using the Poly A Track kit (Promega). Total RNA (20 µg) or 2
µg poly A+ mRNA was electrophoresed through 1%
agarose-formaldehyde gels and transferred to nylon membranes (Nytran,
Schleicher & Schuell, Keene, NH). DNA probes were radiolabeled with
[32P]dCTP (6000 Ci/mmol, Amersham) using the
Oligo-labeling kit (Pharmacia BioTech, Piscataway, NJ) and purified
from unincorporated nucleotides using G-50 spin columns (71).
Hybridization was performed at 65 C using Rapid-Hyb Buffer (Amersham),
and the membranes were washed once with 2x NaCl-sodium citrate
(SSC), 0.1% SDS at room temperature and two times with 0.2x SSC,
0.1% SDS at 65 C before autoradiography. The positions of the 18S and
28S rRNA in total RNA (electrophoresed in parallel for the poly
A+ blots) were used as mol wt standards and were visualized
by EtBr staining before transfer. To determine whether equal amounts of
RNA were loaded and transferred, the membranes were stripped with
boiling 0.1% SDS and rehybridized with a probe for the constitutively
expressed G3PDH mRNA (CLONTECH, Palo Alto, CA).
RT-PCR
DNA-free, total RNA was prepared from the indicated cellular
sources as described for mRNA differential display. RNA (500 ng) was
reverse transcribed at 37 C for 1 h in a 20 µl reaction
consisting of 1x RT buffer (GIBCO), 2.0 mM
dithiothreitol, 0.25 µg of random hexamers (Promega), 2
µM dNTPs, 40 U of RNasin, and 1 µl of MMLV reverse
transcriptase (GIBCO). The cDNA produced from the reaction was divided
into two aliquots and amplified by PCR for 30 cycles at 94 C for 45
sec, 54 C for 45 sec, and 72 C for 1 min using primers designed
specifically for v-myc [5'(CCCAAGGTTGTCATCCTG);
3'(TAATGGGGAGAGACTGGG)] or for ß-actin [5'(ATTGTTACCAACTGGGACG);
3'(TCTCCTGCTCGAAGTCTA)] in 1x PCR buffer (Promega) containing 20
µM dNTPs, 2.0 mM MgCl2, and 1
µl of Taq DNA polymerase (Promega). The PCR products were
resolved and visualized by electrophoresis through 1% agarose gels
containing 0.1 µg/ml EtBr.
Proliferation Assays
Rat AM was obtained from Peninsula Laboratories (Belmont, CA)
and was used at a concentration of 100 nM in media
containing 50 µM isobutylmethylxanthine (IBMX)
(Calbiochem, San Diego, CA) and 0.5 µg/ml insulin (Sigma, St. Louis,
MO) to enhance responsiveness. Swiss 3T3 fibroblasts and 10T1/2
fibroblasts were plated on gelatin-coated dishes in complete medium at
5 x 105 cells/60-mm dish. Forty-eight hours after
plating, the cultures were rendered quiescent with medium containing
0.5% serum (3T3) or 0% serum (10T1/2). After 24 h, parallel
groups were exposed to medium plus 10% serum as a positive control,
serum-depleted media plus AM, IBMX, and insulin or serum-depleted
medium plus IBMX and insulin only. Proliferation was measured for
20 h using reagents and protocols outlined in the
5-bromo-2'-deoxy-uridine (BrdU) Labeling and Detection Kit (Boehringer
Mannheim, Indianapolis, IN). Cultures were treated with a 1:10,000
dilution of DAPI (Hoechst, Somerville, NJ) in PBS (2.7
mM KCl, 1.5 mM KH2PO4,
137 mM NaCl, 15 mM
Na2HPO4, pH 7.4) for 5 min to visulalize all
nuclei. Ten randomly chosen microscope fields (200x) were scored for
BrdU-positive nuclei per total nuclei and averaged. Results are
expressed as a percent relative to proliferation in the positive
control group (10% serum), which is set at 100%. The experiment was
performed three times. Error bars for each group represent the
SEM.
Reporter Gene Assays
10T1/2 cells were transiently transfected using calcium
phosphate DNA precipitation as described previously (7). For luciferase
assays, 300 µl precipitates containing 1 µg of AM-Luc and 0.01,
0.05, 0.1, or 0.5 µg of pMC29 were added to cells seeded 24 h
before transfection in 35-mm multiwell plates at 1 x
105 cells per well. For CAT assays, 400 µl precipitates
were prepared using the amounts of reporter and test DNA indicated in
the figure legends and added to cells seeded 24 h before
transfection at 5 x 105 cells per 100-mm plate.
pEMSVscribe
2 vector DNA (72) was used where necessary to adjust the
total amount of DNA in each precipitate. Five hours after the addition
of precipitates, the cells were shocked osmotically with 20% glycerol
(in serum-free medium) for 2 min, refed complete medium, and maintained
in complete medium for an additional 4050 h. Transfected cells to be
assayed for CAT activity were harvested at 4 C by scraping into 1 ml of
calcium and magnesium-free saline, pH 7.4 (130 mM NaCl, 1.5
mM KH2PO4, 8.0 mM
Na2HPO4, 2.7 mM KCl), pelleted,
washed, resuspended in 230 µl of 0.25 M Tris, 5
mM EDTA, pH 8.0 at 4 C, and sonicated for 20 sec. CAT
assays were performed as previously described (7), and the percent
conversion of [14C]chloramphenicol to the acetylated form
by CAT was determined by scintillation counting. Transfected cells to
be assayed for luciferase activity were scraped, pelleted, washed twice
with PBS, and lysed in 250 µl of 25 mM Tris, pH 7.8, 4
mM EDTA, 1% Triton X-100, 10% glycerol. Luciferase
activity was measured using the Luci-ferase Assay Kit (Promega).
The cell extracts used for the luciferase or CAT assays were normalized
to total protein content (Protein Assay, Bio-Rad, Hercules, CA). All
assays were maintained within the linear range of activity, and each
value reported represents the average of at least four independent
determinations.
Cellular Transformation Assays
The stable transfection of 10T1/2 cells using calcium phosphate
DNA precipitation has been described previously (7, 56). For
transformation assays, two 100-mm plates of cells were transfected with
precipitates containing 200 ng pT24 H-ras and 600 ng pMC29
v-myc together with the indicated amounts of pDCR-rAM.
Twenty-four hours later, the cells were passaged into six 100-mm plates
and maintained in medium supplemented with 5% FBS for approximately 14
days, at which time the cells were fixed and stained with Giemsa, and
the number of foci per plate was determined by visual inspection. The
number of transformed foci generated after the cotransfection of 10T1/2
cells with Ras p21 and v-Myc is set at 100%.
 |
ACKNOWLEDGMENTS
|
---|
The authors acknowledge the generous support of Dr. Frank
Cuttitta (National Cancer Institute, Bethesda, MD) who provided
advice and experimental information (published and unpublished)
pertinent to the topic of this manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Elizabeth J. Taparowsky, Department of Biological Sciences, Purdue University, 1392 Lilly Hall of Life Sciences, West Lafayette, Indiana 47907-1392. E-mail: ejt{at}bilbo.bio.purdue.edu
This work was supported by Grant NP-924 awarded to E.J.T. from the
American Cancer Society and by Postdoctoral Fellowships awarded
to X.W. and M.A.P. from the American Heart Association.
Received for publication March 9, 1998.
Revision received October 20, 1999.
Accepted for publication November 5, 1998.
 |
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