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INTRODUCTION |
The p53 tumor suppressor protein plays an important role in
regulating movement through a number of cell cycle checkpoints (1-3,
38). The activity of p53 is in turn modulated through the action of the
MDM2 protein which is amplified in a variety of tumors (4-6). MDM2 can
associate with p53 (7) and directly inhibit p53's ability to activate
transcription of target genes, such as p21. MDM2 performs this function
in a number of ways. First, the amino-terminal domain MDM2 binds
tightly to the transcriptional transactivation domain of p53 (4-6, 8,
9). Through this interaction, MDM2 blocks the ability of p53 to
activate transcription of specific target genes by repressing the
formation of the preinitiation complex mediated through the TFIIE and
TATA-binding protein subunits of RNA polymerase II (10, 11). The second
blocking activity of MDM2 is that it targets p53 for proteolytic
degradation by the ubiquitin-proteasome pathway (12-14, 40). Third,
MDM2 also functions to shuttle p53 out of the nucleus and into the
cytoplasm, via a nuclear export signal located within the MDM2 protein
(33).
While MDM2 was first identified as a protein that inhibits the action
of p53 it has recently been shown to affect other components of the
G1/S transition. MDM2 was found to associate with both the
E2F1 transcription factor (15) and the retinoblastoma tumor suppressor
(pRb)1 (16). The interaction
with pRb was found to be repressive, in that the association of MDM2
with the carboxyl-terminal domain of pRb inhibited the growth
regulatory function of pRb (16). The interaction with E2F1, over the
amino terminus of MDM2 and the carboxyl terminus of E2F1, was
stimulatory in the sense that MDM2 enhanced the ability of E2F1 to
activate target gene expression (15). Thus MDM2 appears to regulate at
least three of the important players in the G1/S
transition: p53, pRb, and E2F1. It has therefore been concluded that
MDM2 functions in the fashion of a dominantly acting oncogene, driving
cells through the G1/S phase transition, by inhibiting p53
and pRb while helping to enhance the activity of E2F1.
Recently a protein related to MDM2, termed MDMX, has been cloned and
partially characterized (17, 18). The region of highest homology
between MDM2 and MDMX is within the p53-binding domain at the amino
terminus of MDMX. Additionally, a zinc finger domain within the central
portion of MDMX and a ring finger domain at the carboxyl terminus are
conserved between MDMX and MDM2 (17-19). It has been demonstrated that
MDMX associates with p53 and suppresses its activity (17). While MDMX
appears very similar to MDM2, it nonetheless has a number of features
that are distinct. For example, MDMX is not induced by DNA damaging
agents that are known to induce MDM2 (17). This indicates that the MDMX
promoter is likely not transcriptionally transactivated by p53. This is
distinct from MDM2 which is transactivated by p53 (20, 21). Also, MDM2 null mice (both alleles inactivated) are embryonic lethals (22, 23), in
a wild-type p53 background. This indicates that if MDMX is expressed,
it cannot functionally substitute for the absence of MDM2 with regard
to control of p53 activity. However, it should be noted that it has not
yet been demonstrated that MDMX is expressed early in development.
Thus, MDMX likely functions in ways that are separate from MDM2. Yet
the nature of MDMX's role in control of cell proliferation is largely
unknown at this time.
To gain insight into the role of MDMX in control of cell cycle, we have
explored its regulation. We find that the MDMX gene is expressed as
different transcripts. One transcript has a small internal deletion
that leads to the introduction of a stop codon following amino acid
residue 127. This novel transcript encodes only the p53-binding domain
of MDMX and the protein produced is referred to here as MDMX-S. MDMX-S
is better able to suppress p53-mediated transcriptional transactivation
and induction of apoptosis, relative to full-length MDMX. MDMX-S
expression may therefore play an important role in the regulation of
cell proliferation and apoptosis.
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MATERIALS AND METHODS |
Cell Culture, Plasmids, Transfections, and Apoptosis--
All
cell lines were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% bovine calf serum or fetal calf serum. All
transfection experiments were initiated on 50% confluent monolayer
cultures as described by Ausubel et al. (24). Plasmids (a
total of 30 µg) were transfected by the calcium phosphate
procedure (24). The cells were glycerol shocked 5-6 h after DNA addition.
The MDMX and MDMX-S cDNA constructs were cloned into Bluescript KS
just 3' to a FLAG epitope tag sequence. The epitope tag was linked
in-frame to the reading frame of MDMX and MDMX-S. For expression
studies the tagged MDMX and MDMX-S genes were excised from the
Bluescript plasmid and cloned into the eukaryotic expression vector
pRC/CMV (Invitrogen). The wild-type p53 cDNA was cloned into and
expressed from the pcDNA3 expression plasmid (Invitrogen). The
target of p53 was the pRGC
FosLacZ reporter construct containing multiple p53 consensus DNA-binding sites cloned upstream of the basal
Fos promoter (30). As a control, the p
FosLacZ reporter used is
identical to pRGC
FosLacZ, but lacks the p53 consensus sequences
(30). Additionally, the DNA polymerase
promoter and the
dihydrofolate reductase promoter linked to the CAT gene (chloramphenicol acetyltransferase) were used in the co-transfection studies.
Extracts were generated by multiple freeze-thaw cycles approximately
24 h after the glycerol shock. Equal amounts of protein from the
soluble extracts were assayed either for CAT activity by thin layer
chromatography and autoradiography or for
-galactosidase activity by
a color reaction (24). All transfections were performed multiple times.
A plasmid containing the Rous sarcoma virus (RSV)-long terminal repeat
driving expression of the
-galactosidase gene (RSV
-gal) was
included in all transfections of the CAT constructs so that the CAT
activity could be normalized for differences in transfection
efficiency. Additionally, a plasmid containing the SV40 early promoter
linked to CAT was included in all transfections of the
-galactosidase constructs so that the
-gal activity could be
normalized for differences in transfection efficiency.
Apoptosis was measured by transfecting cells with the p53 expression
plasmid, with or without the MDMX/MDMX-S expression plasmids, as is
outlined below. Included on all transfections was the RSV-LacZ reporter
plasmid. At 72 h post-transfection the cells were fixed in 4%
paraformaldehyde, and the
-galactosidase positive cells were
identified using the Beta-Gal Staining Kit from Boehringer Mannheim
(the cells stain blue). The percent of apoptotic cells within the blue
staining population were determined as those with a significantly
rounded morphology and with membrane blebs clearly visible (as in Refs.
34 and 35).
Generation of Extracts and Fusion Proteins--
Extracts for
immunoblots were generated by lysing the cells on ice in 0.1%
Nonidet P-40, 10 mM Tris (pH 7.9), 10 mM
MgCl2, 15 mM NaCl, and the protease inhibitors
phenylmethylsulfonyl fluoride (0.5 mM), pepstatin (2 µg/ml), and leupeptin (1 µg/ml). The soluble fraction was termed
the cytosol. The nuclei were pelleted by centrifugation at 800 × g for 10 min and resuspended in extraction buffer consisting of 0.42 M NaCl, 20 mM Hepes (pH 7.9), 20%
glycerol, phenylmethylsulfonyl fluoride (0.5 mM), pepstatin
(2 µg/ml), and leupeptin (1 µg/ml) for 10 min on ice, then
centrifuged at 14,000 × g for 8 min to pellet the
residual nuclear material. This supernatant fraction was termed nuclear
extract (25).
To generate GST fusion proteins, the MDMX and MDMX-S cDNAs were
cloned into pGEX-5T (Pharmacia) in-frame with the glutathione S-transferase gene (GST). Fusion proteins were produced as
described by Jordan et al. (27). Briefly, the genes were
induced with isopropyl-1-thio-
-D-galactopyranoside for
3 h, bacteria were lysed, and protein was separated from cellular
debris by centrifugation. Using the bacterial extracts, affinity
columns on glutathione-Sepharose (Pharmacia) containing the various
fusion proteins were then generated.
RNA Analysis and RT-PCR Reactions--
Total RNA was isolated
from cells by the Triazol method (Life Technologies, Inc.).
Poly(A)+-enriched RNA was isolated from 400 µg of
confluent NIH3T3 fibroblast total RNA. 1 µg of poly(A)+
RNA was used in the initial RT-PCR reaction. Oligo(dT) was used as the
primer in the reverse transcription reaction which was followed by a
PCR reaction with primers that flank the start and stop codons from the
published sequence of murine MDMX. For the RT-PCR reactions, 1 µg of
total RNA from the various cell lines was used in each reaction with
primers from the murine sequence (the 5' primer, gccctctctatgacatgc
(spanning amino acid residues 96-102) and the 3' primer,
gtcgtgaggtaggcag (spanning amino acid residues 158-163)) and human
sequence (the 5' primer, gccctctctatgatatgc, and the 3' primer,
gctctgaggtaggcag) (17, 18). The primers for the human sequence
represent the same positions as those used for the murine sequence.
These primers flank the site of deletion in MDMX. All total RNAs were
DNase I treated in the RT reaction prior to the PCR reactions.
Additionally, as a control, PCR done in the absence of RT was negative
for any ethidium bromide-stained bands (data not shown).
In Vitro Transcription/Translation--
In vitro
transcription reactions were performed with Bluescript KS plasmids
containing the MDMX and MDMX-S cDNAs. The plasmid constructs were
used directly in the TnT-coupled in vitro
transcription/translation system of Promega, using the nuclease-treated
rabbit reticulocyte lysate, in a total reaction volume of 50 µl.
[35S]Methionine at 0.9 mCi/ml was also included in the reactions.
Western Blot Hybridizations--
Extracts were first
electrophoresed by SDS-PAGE and then the proteins were
electrophoretically transferred onto nitrocellulose, the blots were
washed in TBST buffer (10 mM Tris, pH 8, 150 mM NaCl, 0.05% Tween 20), blocked with 2.5% bovine serum albumin in TBST
for 30 min at room temperature, and then incubated with either the M2
anti-FLAG monoclonal antibody (10 ng/ml, VWR/Kodak/IBI) or polyclonal
antiserum generated against MDMX-S (1:5,000 dilution). The blots were
washed three times (10 min each) with TBST then incubated for 30 min at
room temperature with a 1:7500 dilution of secondary antibody in TBST
(goat anti-mouse or goat anti-rabbit conjugated to alkaline
phosphatase, Vector Labs). The blots were then stained using the
Protoblot system from Promega.
Immunofluorescence--
For indirect immunofluorescence, Calu
cells were plated on 10-cm tissue culture dishes containing glass
coverslips. The cells were transfected with the MDMX or MDMX-S
expression plasmids (or the vector control) and 24 h
post-transfection the plates were washed once in PBS and then fixed
with 4% paraformaldehyde in PBS for 20 min followed by an additional
rinse in PBS. To permeabilize the cells, the coverslips were treated
with PBS plus 0.2% Triton X-100 for 15 min followed by three 5-min
washes in PBS plus 0.2% gelatin, as described by Harlow and Lane (28).
The M2 monoclonal antibody was diluted in PBS plus 0.2% gelatin. 50 µl of diluted antibody was added to a 6-cm culture dish, coverslips
containing the fixed and permeabilized cells were placed cell side down
on the drop of diluted antibody and incubated for 1.5 h at
37 °C (26). The coverslips were washed three times in PBS plus 0.2% gelatin. Fluorescein-conjugated goat anti-mouse IgG (Vector
Laboratories) was diluted to 30 µg/ml in PBS plus 0.2% gelatin.
Again 50 µl of diluted antibody were placed in a dish, the coverslips
placed cell side down and incubated for 30 min at 37 °C. The
coverslips were washed in PBS plus 0.2% gelatin (10 min), PBS plus
0.2% gelatin plus 0.05% Tween 20 (10 min), and finally in PBS (10 min). The nuclei were then stained with the DNA dye
4,6-diamidino-2-phenylindole. The coverslips were rinsed three
times in PBS, once in deionized water, and then they were dried,
mounted, and analyzed by fluorescence microscopy.
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RESULTS |
Identification of A Novel MDMX Transcript Containing an Internal
Deletion--
The MDM2 oncoprotein is a known binding partner of a
number of cellular factors, namely p53, pRb, and E2F1. In our efforts to study the regulation of these key regulatory factors, we initiated work on the recently identified MDM2-related protein, termed MDMX. To
begin studies on MDMX we isolated its cDNA clone by RT-PCR, using
primers that flank the start and stop codons of the published open
reading frame of the murine sequence. We used 1 µg of
poly(A)+ mRNA from confluent nontransformed NIH3T3
fibroblasts in the reaction. It was first reversed transcribed to make
cDNA, then used in the polymerase chain reaction with the primers
that flank the start and stop codons. The RT-PCR products are shown in
Fig. 1. It is apparent from the figure
that two bands are produced in the reaction that are very close in size
to each other (the expected size is 1450 base pairs). The entire
reaction product was then cloned into Bluescript KS+ and a
number of individual isolates were sequenced. While many of the clones
produced the expected published sequence (17) a substantial fraction
had an internal deletion of 68 base pairs. This deleted sequence
results in a change in the reading frame of the protein because it
introduces a new translation stop codon just after the sequence
encoding the p53-binding domain. The deleted sequence is shown in Fig.
2A and the complete reading
frame of this sequence is shown in Fig. 2B. The first 114 amino acid residues are identical to the published sequence and
represent the p53-binding domain (residues 1-100) (17). The change in
reading frame results in an insertion of 13 new amino acids followed by
a stop codon (Fig. 2, A and B). Fig.
2C shows a comparison of full-length MDMX (489 residues) and
the shortened form that we have termed MDMX-S (127 residues). It is
clear that MDMX-S lacks the central and carboxyl-terminal domains
containing the zinc finger and ring fingers, respectively
(17).

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Fig. 1.
PCR of MDMX from NIH3T3 cDNA results in
two bands. Primers flanking the start and stop codons of MDMX were
used in a PCR reaction with 100 ng of cDNA derived from 1 µg of
poly(A)+ mRNA from confluent nontransformed NIH3T3
fibroblasts. The product of the reaction was electrophoresed on a
nondenaturing 10% acrylamide gel. The gel was stained with ethidium
bromide and photographed (the expected size of MDMX is 1450 base
pairs). The position of the markers are shown.
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Fig. 2.
The open reading frame of MDMX-S reveals a
shortened protein. A, the sequence within MDMX that is
deleted in MDMX-S. The brackets define the boundaries of the
DNA sequence in MDMX (top line) that is deleted in MDMX-S
(bottom line). B, amino acid sequence of MDMX-S.
The predicted p53-binding domain that aligns with that of MDM2 is
represented by residues 1-107. The sequence underlined and
in bold is unique to MDMX-S. C, linear alignment
of MDMX and MDMX-S protein sequences. The positions of the p53-binding
domain, the zinc finger, the ring finger, the putative nuclear
localization sequences (NLS) are outlined in MDMX.
D, an RT-PCR reaction was performed on 0.2 and 1.0 µg of
cDNA generated from poly(A)+ selected mRNA from
confluent NIH3T3 fibroblasts using primers that flank the site of the
deletion in MDMX-S. The products of the reaction were electrophoresed
on a 10% polyacrylamide gel. The gel was stained with ethidium bromide
and photographed. The positions of MDMX (201 base pairs), MDMX-S (133 base pairs), and the markers are shown.
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To determine the extent of expression of this new form of MDMX-S, PCR
primers were generated that flank the deleted sequence and PCR was
performed on the original NIH3T3 cDNA pool made from poly(A)+ mRNA from confluent cells. With these primers,
a PCR product corresponding to MDMX would be 201 base pairs in length
while a product corresponding to MDMX-S would be 133 base pairs. As seen in Fig. 2D, two bands of 133 and 201 base pairs are
seen in the polyacrylamide gel from the products of the PCR reaction, indicating that both MDMX and MDMX-S are expressed as transcripts in
these cells.
To ensure that these two bands represent bona fide MDMX and MDMX-S
sequences, the bands were excised from the gel, directly cloned via TA
Cloning (Invitrogen) into the pCR2.1 plasmid vector, and sequenced. DNA
sequence analysis revealed that the 201-base pair band exactly matches
the original MDMX sequence while the 133-base pair band exactly matches
the MDMX-S sequence containing the internal deletion.
It was next important to determine the extent of expression of MDMX and
MDMX-S in other cell lines. Total RNA was isolated from nontransformed
C3H10T1/2 murine embryonic fibroblasts that were growing, confluent or
serum starved for 48 h to arrest in G0/G1
phase of the cell cycle. Total RNA was isolated from the transformed
murine lines psi2 (retrovirally transformed), F9 (teratocarcinoma), and
SP2/0 (myloma/hybridoma). Total RNA was also isolated from nontransformed low passage rat vascular smooth muscle cells (A10 cell
line) that were serum starved or starved and serum stimulated for
8 h. RT-PCR was performed on 1 µg of the total RNA using the primers that flank the site of the deletion. As shown in Fig. 3, A and B, MDMX-S
is expressed as a detectable transcript in all cell lines. In all cell
lines, the levels of MDMX remain somewhat constant. However, in
serum-starved cells, expression of MDMX-S is barely detectable while in
growing cells, and especially those that are transformed, MDMX-S is
easily detectable. Further quantitative methods will be needed to
determine if MDMX-S expression is actually up-regulated in transformed
cells and down-regulated in serum-starved primary cells.

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Fig. 3.
MDMX-S expression is evident in proliferating
and transformed rodent cells. RT-PCR was performed on 1 µg of
total RNA from the indicated mouse cell lines with primers flanking the
deletion in MDMX-S. A, the cells were either growing and
transformed (psi2, F9, SP2/0) or growing, confluent, or serum-starved
nontransformed C3H10T1/2 fibroblasts. B, RT-PCR
of RNA from serum starved or serum starved and 8-h serum stimulated rat
vascular smooth muscle cells (A10 line). The RT-PCR reaction products
were electrophoresed by nondenaturing Tris-borate-EDTA PAGE. The gel
was stained with ethidium bromide. The bands corresponding to MDMX (201 base pairs) and MDMX-S (133 base pairs) are shown. To standardize for
equal loading in the RT-PCR reaction, 1 µg of the total RNA was
simultaneously electrophoresed on a 1% agarose gel, stained with
ethidium bromide, and the position of the 28 S rRNA indicated (below
each RT-PCR gel). Additionally, 1 µg of total RNA was used in an
RT-PCR reaction with primers to glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), shown at bottom of
A and B.
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It was of interest to determine if the expression pattern of MDMX-S in
human cells mirrors that seen in rodent cells. Using the DNA sequence
of human MDMX (18), primers were generated that would flank the
deletion in MDMX-S. These primers were then used in an RT-PCR reaction
with 1 µg of total RNA from the following human/primate cell lines:
COS (SV40 T antigen transformed monkey), HeLa (human cervical
carcinoma), U87Mg (human glioblastoma), 293 (adenovirus transformed
human kidney), Calu-6 (human lung carcinoma), and MCF-7 (human breast
cancer). Also, RNA was isolated from nontransformed 4th passage human
foreskin fibroblasts (HFFs) that were either serum starved or growing.
This is shown in Fig. 4. For comparison, an RT-PCR reaction was also performed on RNA from growing and confluent
NIH3T3 fibroblasts and growing C3H10T1/2 fibroblasts, using the mouse
specific primers, and the products were run on the same gel. MDMX-S is
expressed as a detectable transcript in the RNA from each cell line. In
serum-starved skin fibroblasts, MDMX-S transcripts are not easily
detectable. However, in growing cells, and those that are transformed,
MDMX-S is easily detectable. Further quantitative methods will be
needed to determine if MDMX-S is up-regulated in transformed cells and
down-regulated in low passage cells. In HeLa cells and MCF7 cells,
MDMX-S appears low relative to MDMX. Further experiments will be needed
to determine the basis of this difference.

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Fig. 4.
MDMX-S expression is evident in proliferating
and transformed human cells. Using primers that flank the deletion
in the human MDMX gene, RT-PCR was performed on 1 µg of total RNA
from a variety of human and primate cell lines (right side).
The cells were either transformed (293, Calu, MCF7, HeLa, U87 mg, COS)
or nontransformed HFF's (4th passage). The HFF's were either growing
or were arrested by serum starvation (0.5% for 72 h). The bands
corresponding to MDMX (201 base pairs) and MDMX-S (133 base pairs) are
shown. Also included in the reactions were 1 µg of total RNA from
nontransformed immortalized mouse embryonic fibroblasts (NIH3T3,
C3H10T1/2), either growing or confluent (left side). To
standardize for equal loading in the RT-PCR reaction, 1 µg of total
RNA was simultaneously electrophoresed on a 1% agarose gel, stained
with ethidium bromide, and the position of the 28 S rRNA indicated
(below the RT-PCR gel). Additionally, 1 µg of total RNA was used in
an RT-PCR reaction with primers to glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), shown at the bottom of the
figure.
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Detection of Endogenous MDMX and MDMX-S Proteins in Cell
Extracts--
To determine if endogenous MDMX and MDMX-S protein
expression could be detected in cell extracts, rabbit polyclonal
antibodies were generated against a GST fusion with MDMX-S. These
antibodies should recognize both MDMX and MDMX-S. Two separately
generated antisera were used in immunoblots with 50 µg of nuclear and
cytosolic extract from COS cells. COS cell extracts were used because
they appeared to express high levels of the MDMX-S transcript (Fig. 4).
As shown in Fig. 5A, antisera
from one rabbit recognizes predominantly MDMX-S present in only the
nuclear extract. Its size of 27 kDa is substantially higher than its
predicted molecular mass of 15 kDa, suggesting that the protein
undergoes some modification. The other antisera strongly recognizes
MDMX, as shown in Fig. 5B, and weakly recognizes MDMX-S.
Thus, both MDMX and MDMX-S appear to be expressed as detectable gene
products in COS cells.

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Fig. 5.
Endogenous MDMX and MDMX-S are detectable in
COS cell extracts by immunoblot, using polyclonal antiserum to
MDMX-S. 50 µg of COS nuclear and cytosolic extract were
electrophoresed by SDS-PAGE and blotted onto nylon membrane. Identical
blots were generated and incubated with the different antisera
(A, sera number 234; B, sera number 235)
generated against MDMX-S. Identical blots were also probed with each
preimmune sera. Following incubation with the primary antibody, the
blots were incubated with a secondary antibody coupled to alkaline
phosphatase. Shown are the developed blots. The bands corresponding to
MDMX and MDMX-S are indicated.
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Detection of an MDMX-S Protein following Translation in Vitro and
following Ectopic Expression in COS Cells--
To begin functional
studies of MDMX-S, the FLAG epitope tag was cloned at the 5' end of the
MDMX-S gene (at the amino terminus of the protein). This tagged MDMX-S
gene was then transcribed and translated in vitro using a
rabbit reticulocyte lysate, with [35S]methionine/cysteine. The products of the reaction
were analyzed by SDS-PAGE, the gel immunoblotted, and the blot probed
with the M2 antibody which recognizes the epitope tag. Two protein
bands of 17 and 27 kDa were detected in the immunoblot, as shown on the
right side of Fig. 6 ("Western").
When the blot was exposed to x-ray film these bands were also detected
by autoradiography, as seen on the left side of Fig. 6
("35S label"). If the 17-kDa form represents the
correct size MDMX-S protein as predicted from the open reading
frame, then the 27-kDa form is possibly a modified version. We do
not yet know the nature of any potential modification although MDMX-S
is rich in serine, a potential site for phosphorylation.

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Fig. 6.
Coupled in vitro
transcription/translation of MDMX-S reveals two protein
bands. The MDMX-S protein was radiolabeled by coupled in
vitro transcription/translation (TnT System, Promega). The
resulting [35S]Met/Cys radiolabeled protein was separated
by SDS-PAGE followed by immunoblotting. The blot was incubated with the
M2 antibody directed against the epitope tag followed by incubation
with a secondary antibody coupled to alkaline phosphatase. Shown on the
right is the developed blot. The blot was then exposed to
x-ray film for autoradiography (the film is shown on the
left). The two protein bands of 17 and 27 kDa produced by
MDMX-S are indicated.
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The epitope-tagged MDMX and MDMX-S genes were then cloned into the
pRC/CMV expression plasmid for use in transfection studies. The
plasmids were transfected into COS cells and nuclear and cytosolic extracts were generated at 48 h post-transfection. The extracts (50 µg) were electrophoresed by SDS-PAGE, immunoblotted, and
incubated first with the M2 monoclonal antibody and then with a
secondary antibody conjugated with alkaline phosphatase (shown in Fig.
7A). It is clear that
expression of both proteins can be detected in the extracts, and that
by the intensity of staining, both proteins are expressed to roughly
equal levels. The distribution of protein between nuclear and cytosolic
extracts may be due to overexpression. It is also clear that the
ectopically expressed MDMX-S is approximately 27 kDa, equivalent in
size to the larger species seen in the in vitro
translation reactions from Fig. 6.

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Fig. 7.
Ectopically expressed MDMX and
MDMX-S are detectable in COS cell extracts by immunoblot and in Calu
cells by indirect immunofluorescence. A, COS cells were
transfected with pRC/CMV vector alone ("control") (10 µg),
pRC/CMV-MDMX (10 µg), or pRC/CMV-MDMX-S (10 µg). Forty-eight hours
post-transfection, the cells were lysed and extracts generated. 50 µg
of COS nuclear and cytosolic extract were electrophoresed by SDS-PAGE
and blotted onto nylon membrane. Blots were generated and incubated
with the M2 antisera specific to the epitope tag. This was followed by
a secondary antibody coupled to alkaline phosphatase. Shown are the
developed blots. The bands corresponding to MDMX and MDMX-S are
indicated. B, Calu cells grown on coverslips were
transfected with pRC/CMV-MDMX (10 µg), pRC/CMV-MDMX-S (10 µg), or
with the vector alone (10 µg). Forty-eight hours post-transfection,
the cells were fixed and processed for indirect immunofluorescence (M2
primary antibody directed against the epitope tag and a fluorescein
isothiocyanate conjugated secondary antibody). Shown in the figure are
two representative fluorescent positive cells for each transfection.
Fluorescein isothiocyanate fluorescence levels per cell were
quantitated using the Bio-Rad COMOS software program (Bio-Rad
Microsciences Division). The relative fluorescence intensity is an
average of 40 positively fluorescing cells (MDMX and MDMX-S) or 40 nonfluorescing cells (vector control).
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Similar transfection experiments were then performed in Calu cells.
Unfortunately the ectopically expressed MDMX and MDMX-S could not be
detected by immunoblot. Expression could be detected by indirect
immunofluorescence, however, as shown in Fig. 7B (the secondary antibody was conjugated to fluorescein isothiocyanate). The
control transfected cells (vector only) were completely negative for
fluorescein isothiocyanate fluorecence (data not shown). To determine
if the level of expression was equal between MDMX and MDMX-S, the
fluorescence intensity of a number of fluorescent positive cells was
quantitated using a confocal microscope and the Bio-Rad COMOS software
program (as in Ref. 41). For each transfection, about 40 individual
cells were image analyzed and the relative level of fluorescence
intensity measured. Shown in the lower part of Fig.
7B are the results of this quantitation. It is clear that
MDMX and MDMX-S protein levels are nearly identical. Thus the data from
Fig. 7, A and B, indicate that MDMX and MDMX-S are expressed to equal levels in both COS and Calu cells.
MDMX-S Is a More Potent Repressor of p53-mediated Transcription
Than MDMX--
To test the function of MDMX-S, its role in
p53-mediated transcriptional activation was assessed. MDMX-S and MDMX,
cloned into the pRC/CMV expression vector, were used in transfection studies along with a p53 expression plasmid and a p53 target promoter containing multiple p53 consensus binding sites (pRGC
FOS-LacZ) (30).
Transfections were first performed in COS cells and promoter activity
was monitored at 24 h post-transfection. As shown in Fig.
8A, left panel, p53 expression
is able to significantly up-regulate transcription from this promoter,
to approximately 17-fold. This is consistent with the ability of p53 to
enhance expression from this promoter. Coexpression of MDMX reduces the
level of p53-mediated transcription to 3-fold above the control,
consistent with its role in repressing p53 function (17).
Interestingly, MDMX-S was able to reduce the level of p53-mediated
transcription much further, to only 50% of the control. This is 6-fold
greater than was accomplished by MDMX.

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Fig. 8.
MDMX-S is a potent suppressor of p53-mediated
transcriptional activation. COS cells (A) and Calu
cells (B) were transfected with pRGC FOS-lacZ vector (10 µg) in the presence or absence of a p53 expression plasmid (1 µg)
(left panels). Additionally, where indicated, the cells were
co-transfected with pcDNA3-MDMX (10 µg) or pcDNA3-MDMX-S
(10 µg). Twenty-four hours post-transfection, the cells were
lysed and extracts generated. Ten µg of extract was used in an assay
for -galactosidase activity. Shown are the relative levels of
activity over multiple experiments. On the right side of
A and B is the data showing similar transfections
with a control promoter lacking the p53 consensus binding sites
(p FOS-LacZ) (10 µg). C, Calu cells were transfected
with a pDHFR-CAT vector (10 µg) (left) or a DNA polymerase
-CAT vector (10 µg) (right) in the presence or absence
of a p53 expression plasmid (1 µg). Additionally, where indicated,
the cells were co-transfected with pcDNA3-MDMX (10 µg) or
pcDNA3-MDMX-S (10 µg). Twenty-four hours post-transfection, the cells
were lysed and extracts generated. Twenty µg of extract was used in
an assay for CAT activity. Shown are the relative levels of activity
over multiple experiments.
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When transfections were next performed in Calu cells, which are devoid
of a functional p53, a more striking effect was seen. Expression of
MDMX reduced the level of p53-mediated transcription by one-half.
MDMX-S on the other hand was able to reduce the level of p53-mediated
transcription to background levels which is 12-fold more than was
accomplished by MDMX (Fig. 8B, left panel). Taken together,
it therefore appears that MDMX-S is a much more potent repressor of
p53-mediated transcriptional activation than full-length MDMX.
As controls in these experiments, the p
FosLacZ reporter construct,
lacking the p53-binding sites, was used in similar transfections. As
shown in Fig. 8, A and B, right
panels, p53 is not able to activate transcription from this
promoter. Furthermore, MDMX-S and MDMX have no additional effect on
transcription from p
FosLacZ. This indicates that the effect of
MDMX-S on the pRGC
FOS-LacZ promoter plasmid, containing the
p53-binding sites, is dependent on p53. As additional controls in these
experiments, MDMX alone or MDMX-S alone had no effect on transcription
from either of the target promoters (data not shown).
The biological effect of p53 is to induce both cell cycle arrest in
G1 phase and apoptosis. A G1 phase arrest would
be evident as a suppression of S phase gene expression, represented,
for example, by dihydrofolate reductase (DHFR) and DNA polymerase
(DNA pol
). Thus, overexpression of p53 should down-regulate expression from the DHFR and DNA pol
promoters. This is shown in
Fig. 8C, where coexpression in Calu cells of a DHFR
promoter-CAT construct or a DNA pol
promoter-CAT construct, along
with a p53 expression plasmid results in a significant drop in
transcription, compared with the activity of these promoters in the
absence of p53. Promoter activity was monitored at 24 h
post-transfection when the levels of apoptosis were low. Additionally,
RSV-LacZ was co-transfected so that the levels of CAT activity could be corrected for any differences in transfection efficiency or induction of apoptosis. As seen in Fig. 8C, when MDMX-S is coexpressed
along with p53, the inhibitory effects of this tumor suppressor are completely abolished, resulting in full or even enhanced promoter activity. Coexpression of MDMX on the other hand only partially restores activity from these promoters. These data are consistent with
the results presented above that MDMX-S is a more potent inhibitor of
p53 activity than MDMX. Note that throughout these experiments, as in
Fig. 8, A-C, MDM2 was found to suppress p53-mediated transcription to the same extent as full-length MDMX (data not shown),
similar to what has been previously described (17, 18).
MDMX-S Inhibits p53-mediated Apoptosis--
The role of MDMX-S in
p53-mediated apoptosis was next assessed. Evidence indicates that p53
mediates apoptosis in part through its transcriptional activation
domain (37), which is the region likely bound by MDMX and MDMX-S. Thus
overexpression of MDMX and MDMX-S should block p53-induced apoptosis.
The MDMX-S and MDMX expression plasmids were therefore used in
transfection studies along with a p53 expression plasmid and RSV-LacZ.
Transfections were performed in COS cells and in Calu cells.
-Galactosidase positive cells were identified by staining and the
percent apoptotic cells within this
-gal positive population was
determined. As shown in Fig. 9, p53
expression results in a significant increase apoptosis, at 72 h
post-transfection, in both cell types. This is consistent with the
known ability of p53 to induce apoptosis when overexpressed
(1-3, 38). Coexpression of MDMX-S reduces p53-mediated
apoptosis by 62% in both Calu cells and COS cells. This is
consistent with its ability to strongly suppress p53 transcriptional activity as described above. MDMX coexpression, however, was only able
to reduce the level of p53 apoptosis by 32-34% in these two cell
types. Therefore, it appears that MDMX-S is a more potent repressor of
p53-mediated apoptosis than full-length MDMX.

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Fig. 9.
MDMX-S represses p53-mediated apoptosis to a
greater extent than MDMX. Calu cells (right panel) and
COS cells (left panel) were transfected with a p53
expression vector (10 µg), in the presence or absence of the MDMX or
MDMX-S expression vectors (10 µg). Also included in each transfection
was the RSV-LacZ expression plasmid (1 µg). At 72 h
post-transfection, the cells were fixed on the plates and the
-galactosidase positive cells were identified by staining. An
apoptotic cell was identified as a rounded cell, detaching from the
plate showing membrane blebbing characteristic of apoptosis
(34-36). Shown are the percent of apoptotic cells within the
population of -galactosidase positive cells.
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Association of p53 with MDMX and MDMX-S--
Binding experiments
were next performed to determine if wild-type p53 could associate with
both the MDMX and MDMX-S proteins. p53 was radiolabeled in an in
vitro transcription/translation reaction and then equal quantities
of the labeled p53 were applied to small batch columns (100 µl)
containing GST, GST-MDMX, or GST-MDMX-S. It is estimated that nanogram
quantities of p53 are applied to the columns which contain microgram
quantities of the fusion proteins (thus the fusions are in
1,000-10,000-fold excess). The columns were washed extensively with
buffer and then the bound material was eluted by boiling the columns in
SDS-PAGE sample buffer. The eluate was electrophoresed by SDS-PAGE and
the gel exposed to film. As shown in Fig.
10A, it is clear that p53
associates with both MDMX and MDMX-S but does not associate with GST
alone. This is consistent with the notion that the amino-terminal
domain of MDMX is the p53-binding domain (17, 18). However, it appears from the film that the affinity of p53 for MDMX-S may be higher than
for MDMX. In these experiments we consistently see an approximate 8-fold increase in the amount of p53 bound to MDMX-S, compared with
MDMX. Fig. 10B shows the Coomassie Blue-stained gel of the fusion proteins from the affinity columns.

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Fig. 10.
Wild-type p53 associates with MDMX-S, while
a mutant p53 does not. A, the p53 protein was
radiolabeled by coupled in vitro transcription/translation.
The resulting [35S]Met/Cys radiolabeled protein was
separately applied to small batch affinity columns containing GST,
GST-MDMX, and GST-MDMX-S. The columns were washed extensively in buffer
and then boiled directly in SDS-PAGE sample buffer and electrophoresed
by SDS-PAGE. The gel was dried and then exposed to x-ray film for
autoradiography (the film is shown). The input p53 protein is also
shown at one-fifth the amount loaded onto the columns. B,
the GST fusion columns were identified by staining of the gel with
Coomassie Blue. Shown are GST alone, GST-MDMX, and GST-MDMX-S.
C, the mutant p53(L22Q,W23S) protein was radiolabeled by
coupled in vitro transcription/translation. The resulting
[35S]Met/Cys radiolabeled protein was separately applied
to small batch affinity columns containing GST and GST-MDMX-S. The
columns were washed extensively in buffer and then boiled directly in
SDS-PAGE sample buffer and electrophoresed by SDS-PAGE. The gel was
dried and then exposed to x-ray film for autoradiography (the film is
shown). The input mutant p53(L22Q,W23S) protein is also shown.
D, the GST fusion columns were identified by staining of the
gel with Coomassie Blue. Shown are GST alone and GST-MDMX-S.
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As a control for these experiments a mutant p53 was used that fails to
associate with MDM2 (39). This mutant has changes at residues 22 and 23 (Leu22 to Gln and Trp23 to Ser) that result in
an abrogation of MDM2 binding (39). As described above, p53(L22Q,W23S)
was radiolabeled in an in vitro transcription/translation
reaction. Equal quantities of radiolabeled protein were then applied to
small batch columns (100 µl) containing either GST or GST-MDMX-S. The
columns were washed extensively with buffer and then the bound material
was eluted by boiling the columns in SDS-PAGE sample buffer. The
eluate was electrophoresed by SDS-PAGE and the gel exposed to film.
Shown in Fig. 10C, it is clear that p53(L22Q,W23S) does not
associate with MDMX-S. This is consistent with the notion that residues
Leu22 and Trp23 within p53 are essential for
binding both MDM2 and MDMX. Fig. 10D shows the Coomassie
Blue-stained gel of the fusion proteins from the affinity columns.
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DISCUSSION |
Here we have identified a novel transcript of the MDMX gene. This
form of MDMX, termed MDMX-S, produces a shortened protein comprised of
the p53-binding domain of MDMX and lacking the central and
carboxyl-terminal regions. This truncation is due to an internal deletion of 68 base pairs within the normal MDMX transcript that leads
to the introduction of a stop codon just after the sequence encoding
the p53-binding domain at residue 127. This deletion, which may be due
to some form of regulated alternative splicing, also changes the
reading frame and introduces 13 new amino acids into the
carboxyl-terminal end of the truncation. At this time it is not known
what role these new residues play in MDMX-S function.
What is perhaps most intriguing about the different forms of MDMX
mRNA is that the MDMX-S transcript is expressed in a variety of
mouse and human transformed cell lines. In nontransformed quiescent cells such as serum-starved murine embyonic fibroblasts, HFFs, and rat
vascular smooth muscle cells, MDMX-S transcripts are difficult to
detect by RT-PCR (especially in the low passage HFF and rat cell
lines). In proliferating cells, and especially in some of the
transformed cells, MDMX-S transcripts are easily detectable. It should
be noted that quantitative methods will be needed to determine the
exact levels of MDMX versus MDMX-S. That MDMX-S is expressed
in proliferating and transformed cells suggests that it may play an
important role in the control of cell proliferation.
Examination of the MDMX-S protein reveals that it binds p53 strongly,
and with what might be a higher affinity than the binding of MDMX to
p53. This finding appears consistent with the biological activity of
MDMX-S when it is ectopically expressed in a variety of cell lines. In
transient transfection studies, MDMX-S was better able to suppress
p53-mediated gene expression, when compared with MDMX. This was shown
using a promoter containing multiple p53-binding sites that is able to
be transactivated by p53. In a second set of experiments, p53
overexpression was able to down-regulate transcription from the
promoters of the cell cycle control genes dihydrofolate reductase and
DNA polymerase
. This was likely due to a G1 phase cell
cycle arrest or a general repressive effect of wild-type p53
expression. This down-regulation was completely reversed by MDMX-S,
while MDMX was not nearly as effective. Finally, MDMX-S was able to
block p53-mediated induction of apoptosis to a greater extent than
MDMX. These data indicate that MDMX-S blocks the
transcriptional/apoptotic effects of p53 to a greater extent than is
accomplished by MDMX. Thus, the expression of MDMX-S in transformed
cells may represent an important step in neoplastic transformation
possibly because it may be more effective at blocking p53 function than
full-length MDMX.
Since MDMX-S lacks the central and carboxyl-terminal zinc finger and
ring finger domains, it may be under unique regulation. For example,
the central and carboxyl-terminal regions may be important for
protein-protein interaction, a newly described role for zinc and ring
finger motifs (19). In our preliminary studies, MDMX does not appear to
bind DNA and hence the zinc/ring fingers do not appear to contribute to
that function. The central and carboxyl-terminal domains may aid in
targeting MDMX for proteosome-mediated degradation via ubiquitin ligase
E3, as appears to be the case for MDM2 (12-14, 29, 40). Given its
differences with MDMX, MDMX-S will therefore likely have affects on
cell cycle control that are distinct from that of MDMX.
That the MDMX gene is expressed as multiple transcripts is similar to
the expression pattern of MDM2, which is expressed as multiple
mRNAs, each with varying translation start and stop codons (31,
32). These multiple transcripts produce different sized MDM2 proteins
(31, 32). MDM2 and MDMX appear to share some features of their gene
expression patterns, in that a variety of proteins can be produced from
expression of a single gene. This form of regulation of gene expression
may be a way to expand the functional capabilities in the MDM2/MDMX
gene family.
Another feature of the studies presented here is that MDMX, MDMX-S, and
MDM2 migrate on SDS-PAGE significantly higher than their predicted
molecular weights. The fact that in vitro translated MDMX-S
migrates at approximately 17 and 27 kDa would indicate that the 27-kDa
protein may be a modified form of MDMX-S. Given that MDMX-S contains 14 serines and 5 threonines, it is possible that it is modified by
phosphorylation. Since both MDMX-S and MDMX migrate at approximately
10-12 kDa above their predicted values, modification at the amino
terminus may be the reason why full-length MDMX migrates in SDS-PAGE
higher than its predicted molecular weight. Current efforts are
underway to determine if this is the case.