From the Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago, Chicago, Illinois 60612
Received for publication, May 31, 2002, and in revised form, September 10, 2002
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ABSTRACT |
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PAX3-FKHR is an oncogenic form of the
developmental regulator Pax3 transcription factor. PAX3-FKHR results
from a t(2,13) chromosomal translocation, a unique genetic
marker of alveolar rhabdomyosarcoma. In this study, we showed that
ectopic expression of PAX3-FKHR, but not Pax3, in fibroblasts altered
cell cycle control and accelerated G0/G1
to S cell cycle transition. PAX3-FKHR-expressing cells had reduced
expression of p27Kip1 protein, a key cell cycle regulator.
The reduction in p27Kip1 levels by PAX3-FKHR resulted from
destabilization of p27Kip1 as shown by cycloheximide
treatment and in vivo pulse-chase labeling experiments. The
reduced p27Kip1 protein level in PAX3-FKHR-expressing cells
was restored to the level of control cells by treatment with chemical
inhibitors that specifically blocked 26 S proteasome activity. Along
with the reduction in p27Kip1 protein, PAX3-FKHR-expressing
cells exhibited elevated expression of F-box Skp2 protein, a
substrate-specific component of SCF (Skp1-Cullin-F box protein) ligase
involved in the cell cycle-dependent control of
p27Kip1 ubiquitination and 26 S proteasome dependent
degradation. Finally, we showed that ectopic expression of
p27Kip1 in PAX3-FKHR-expressing cells significantly reduced
the proliferation and colony-forming potential of these cells,
implicating that down-regulation of p27Kip1 protein played
an active role in the PAX3-FKHR-directed cell transformation.
Rhabdomyosarcomas are the most common soft-tissue sarcoma of
childhood (1-3). These are heterogeneous groups of malignant skeletal
muscle tumors with alveolar rhabdomyosarcoma
(aRMS)1 as the more malignant
subtype (4-8). Most of the alveolar tumors carry a characteristic
t(2,13)(q35;q14) chromosomal translocation (9, 10), and a minor group
of aRMS carries a variant t(1,13)(p36;q14) chromosomal translocation
(11). Both chromosomal translocations result in the same in-frame
fusion of Pax genes (Pax3 gene from chromosome 2 and Pax7 gene from chromosome 1) to the FKHR gene located on chromosome 13 (11-13).
Pax3 and Pax7 genes belong to a nine-member gene
Pax transcription factor gene family (14). All
Pax gene members share a common 128-amino acid DNA binding
domain termed the paired box domain. Many members of the Pax family,
including Pax3 and Pax7, contain a second DNA
binding domain of the homeodomain class. The Pax3 and Pax7
transcription activation domain is localized within a serine-,
glycine-, and threonine-rich region at the COOH terminus. Both
Pax3 and Pax7 genes have been shown to play
critical roles in the development of myogenic cell lineage (15-17).
FKHR gene is one of the three FKHR transcription
factor gene family members (FKHR, FKHRL1, AFX) that are
closely related to the HNF3 gene family (18-21).
FKHR protein is characterized by an NH2-terminal winged-helix DNA binding domain and a COOH-terminal proline-rich acidic
transcriptional activation domain. The FKHR family, like the
Pax family, has also been implicated in developmental
regulation (22, 23). Recently, the FKHR family members have
been indicated to play a role in IGF-dependent cell
survival (24, 25).
The aRMS-associated chromosomal translocations join the paired and
homeodomain DNA binding motifs of the Pax3 and
Pax7 genes to the bisected DNA binding domain and
COOH-terminal transactivation domain of FKHR gene, leading
to the expression of PAX3-FKHR and PAX7-FKHR fusion transcription
factors, respectively (11-13). Much information on the oncogenic
potential of the PAX-FKHR fusion proteins has come from studies that
compare the functional differences between PAX3-FKHR and wild type Pax3
proteins since the bisected DNA binding domain of FKHR has not been
shown to bind DNA. These studies have revealed two major differences in
the activity of the fusion protein as compared with wild type
counterpart. One, PAX3-FKHR is a much more potent transcription
activator than the wild type Pax3 toward Pax3-responsive DNA elements
(26), and second, PAX3-FKHR is able to target gene sequences that are
not normally regulated by the wild type Pax3 (27). The increased transcription potency in PAX3-FKHR is thought to result from the inability of the Pax3 NH2-terminal cis-acting repressor
domain and of the trans-acting repressor proteins that interact with Pax3 DNA binding domains to repress the FKHR transactivation domain (28-30). The increased gene-targeting range in PAX3-FKHR is proposed to result from uncoupling of the functional interdependency
between the paired and homeodomain DNA binding motifs (31). The
transactivation activity of the wild type Pax3 depends on the
cooperative interaction between the two DNA binding motifs. As a result
of fusion process, PAX3-FKHR is now able to bind and transactivate DNA
sequences through its homeodomain alone. Recent reports indicate that
the homeodomain, but not the paired domain, is critical for its
cellular transformation function (31-33), suggesting that the
importance of identifying those genes whose expression/or function is
selectively disrupted by the oncogenic form of Pax3 in understanding
the mechanistic links between PAX3-FKHR and oncogenic transformation
and, ultimately, the pathogenesis of rhabdomyosarcoma.
In this study, we present evidence to show that the oncogenic form of
Pax3 disrupts cell cycle regulation in fibroblast cells by negatively
regulating the expression of p27Kip1 protein. The
p27Kip1 is a member of the CIP-KIP family of cell cycle
regulators (34, 35). The level of p27Kip1 protein increases
when cells exit the cell cycle in response to signals such as serum
deprivation, exposure to growth inhibiting and differentiation factors
(36-38), and loss of adhesion to extracellular matrix (39-43). By
contrast, p27Kip1 levels decline dramatically in late
G1, relieving the inhibition of cyclin E- and cyclin
A-Cdk2 activities, thus promoting G1/S-transition and S-phase progression. The concentration of p27Kip1
protein in cells is regulated at multiple levels including
transcription (44, 45), translation (46-48), and post-translational
mechanisms (49-52). The present study provides evidence that one
mechanism involved in PAX3-FKHR transformation is through a decrease in p27Kip1 stability due to increased action of Skp2-mediated
26 S proteasome degradation.
Reagents--
Constructs containing mouse-human PAX3-FKHR hybrid
cDNA and mouse Pax3 cDNA have been previously described (27,
53). The retrovirus-producing Phoenix cell line was obtained from Dr.
Gary Dolan. The constructs containing wild type mouse
p27Kip1 cDNA, human mutant p27Kip1
cDNA, and human p45Skp2 were gifts from Dr. Hiroshi
Kyokawa (Department of Molecular Genetics, University of Illinois at
Chicago), Dr. Kei-ichi Nakayama (Medical Institute of Bioregulation,
Kyushu University, Fukuoka, Japan), and Dr. Hideyo Yasuda (Tokyo
University of Pharmacy and Life Sciences, Tokyo, Japan),
respectively. Antibodies against p27Kip1 (C-19),
p45Skp2 (H-435), cyclin A (H-432), cyclin E (M-20), cyclin
D (C-20), cyclin B1 (H-433), Cdk2 (H-298), p16 (F-12),
p19Skp1 (C-20) were from Santa Cruz; antibody against
Cullin1 was from NeoMarker; antibody against Rb was from
Pharmingen; antibody against Pax3 (ab-2) was from Geneka; antibody
against Cell Culture--
Murine NIH3T3 fibroblast cell line and its
derivative cell lines were maintained in Dulbecco's modified Eagle's
high glucose medium supplemented with 200 units/ml penicillin, 50 µg/ml streptomycin, 1 mM glutamine, and 10% (v/v) bovine
calf serum. Human aRMS cell lines RH4 and RH30 were maintained in 10%
(v/v) fetal calf serum-supplemented Dulbecco's modified Eagle's
medium. NIH3T3 cells stably expressing vector, Pax3, or PAX3-FKHR
cDNA were established by the retroviral infection method as
previously described (31). All experiments were repeated, and results
were confirmed using both clonal cell lines and the pooled cell
population. In this report, with the exception of Fig. 9, only data
obtained from pooled cells are presented.
Cell Extract Preparation--
Whole cell extract was prepared
from cells by either lysing cells in high salt extraction buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM
EDTA, 0.1% Nonidet P-40, 25% glycerol, 1 mM DTT, 0.5 mM orthovanadate, 0.5 mM phenylmethylsulfonyl
fluoride, 2 µg/ml mixture of leupeptin, pepstatin A, and aprotinin)
or in radioimmune precipitation assay buffer (20 mM Tris,
pH 8.0, 137 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM DTT, 2 µg/ml mixture of aprotinin, leupeptin, and
pepstatin A, 0.5 mM orthovanadate, 1 mM
phenylmethylsulfonyl fluoride) for 30-60 min at 4 °C with agitation. Extract was then cleared of cell debris by brief
centrifugation, and clarified supernatant was stored at Western Blot Analysis--
Extract was size-fractionated on an
SDS-polyacrylamide gel and transferred electrophoretically onto
nitrocellulose membrane in buffer containing 20 mM Tris, pH
8.0, 192 mM glycine, and 20% methanol. Proteins of
interest were detected with specific antibody by the chemiluminescent
antibody detection kit (PerkinElmer Life Sciences) under application
conditions recommended by the manufacturer.
RNA Preparation and Northern Blot Analysis--
Total RNA was
prepared from cultured cells by the Trizol extraction method
(Invitrogen) according to the manufacturer's recommendations. 15 µg
of total RNA isolated from cells was denatured, electrophoresed on a
formaldehyde-style gel, and blotted to nitrocellulose membrane. The
membrane was hybridized to nick-translated DNA probe. Afterward, the
membrane was washed twice at room temperature at low stringency (2×
SSC (1× SSC = 0.15 M NaCl and 0.015 M
sodium citrate) and 0.1% SDS) and then twice at 42 °C at
high stringency (0.2× SSC and 0.1% SDS). The washed membrane was
exposed for autoradiography at Cyclin-associated Cdk2 Kinase Assay--
In brief, cells were
first rinsed twice with ice-cold phosphate-buffered saline and then
lysed in immunoprecipitation buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM
EGTA, 10% glycerol, 1 mM DTT, 0.1% Tween 20, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 M
orthovanadate, 1 mM NaF, 2 µg/ml mixture of leupeptin, aprotinin, and pepstatin A.). Cell lysate was cleared with debris by
brief centrifugation. For immunoprecipitation, extract was first
pre-cleared with protein A-coated Sepharose beads at 4 °C for 1 h and then used in incubation with either cyclin A- or cyclin E-specific antibody for 2 h at 4 °C followed by protein
A-Sepharose treatment for 1 h at 4 °C. At the end of the
immunoprecipitation reaction, the beads were thoroughly washed 3-4
times with immunoprecipitation buffer and twice with kinase buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM DTT, 10 mM E2F Electrophoretic Mobility Shift Assay--
Electrophoretic
mobility shift assay was performed by incubating whole cell high salt
extracts (10 µg) with nonspecific salmon sperm DNA (0.5 mg/ml) in a
binding buffer containing 20 mM HEPES, pH 7.4, 0.5 mM EDTA, 10% glycerol, 5 mM DTT, 1 mM MgCl2, and 50 mM KCl for 5 min
on ice. Routinely, 0.2 ng of a 32P-labeled E2F DNA probe
prepared by Klenow labeling was added to the electrophoretic mobility
shift assay reaction mixture (final volume of 20 µl) and allowed to
form DNA-protein complexes during a 20-min incubation at room
temperature. The complex was analyzed on a 5% nondenaturing
polyacrylamide gel. Electrophoresis was carried out in 0.25× Tris
borate-EDTA buffer at 120 V at 4 °C until the bromphenol blue dye
reached the bottom, and the gel was dried and autoradiographed.
In Vitro Protein Degradation Assay--
The in vitro
p27Kip1 degradation reaction was set up as previously
described (54). In vitro degradation reaction was set up by
incubating 25-100 µg of hypotonic cell extract with 0.3 fmol of
35S-labeled in vitro translated
p27Kip1 in the presence of ATP-regenerating system (1 mM ATP, 10 µg/ml creatine kinase, 20 mM
creatine phosphate) and 1/25 (v/v) rabbit reticulocyte lysate at
30 °C for the indicated times. The reaction was terminated by the
addition of Laemmli sample buffer, analyzed by SDS-PAGE, and autoradiographed.
Inactivation of PAX3-FKHR-mediated activity in rhabdomyosarcoma
cells significantly impairs the growth of the tumor cells, implying
that the fusion protein actively participates in the pathological
progression of the disease (55). Previous studies clearly demonstrate
that PAX3-FKHR is a dominant oncoprotein in that it is sufficient to
induce transformation in primary fibroblast cells (56) and immortalized
murine embryonic fibroblast cells (31, 33); however, the mechanisms and
pathways involved in PAX3-FKHR-initiated transformation have not been
characterized. In this report, we focused on examining the effect of
PAX3-FKHR on cell cycle control using NIH3T3 embryonic fibroblast cell
line as a study model.
PAX3-FKHR Promotes G0/G1 to S
Transition of Cell Cycle in NIH3T3 Fibroblast Cells--
In our
previous studies, we have shown that ectopic expression of Pax3
protein, even at a level severalfold higher than PAX3-FKHR, is unable
to transform NIH3T3 cells (31). To better understand the changes in
growth caused by PAX3-FKHR transformation, we examined the effect of
PAX3-FKHR on cell proliferation using NIH3T3 cells that stably
expressed PAX3-FKHR. As controls we used NIH3T3 cells that were stably
transfected with the expression vector or PAX3 expression vector. As
shown in Fig. 1, Pax3-expressing cells
displayed a comparable growth rate as the control cells, whereas
PAX3-FKHR-expressing cells exhibited a greater proliferation rate than
control cells.
PAX3-FKHR Overexpression Results in Reduced Expression of Cyclin
Kinase Inhibitor, p27Kip1--
To assess the effect of
PAX3-FKHR on cell cycle behavior, we investigated the expression
pattern of a number of key cell cycle regulatory proteins by Western
blot analysis (Fig. 2). Although there
were subtle variations in a few cell cycle regulators such as cyclin D1
and cyclin A, the most notable and reproducible changes were the
decreased level of cyclin kinase inhibitor p27Kip1 protein
and the increased level of hyperphosphorylated form of Rb. To verify
that our observation with the p27Kip1 down-regulation was
not a consequence of selecting the fastest-growing cells during
establishment of stable cell lines, we carried out additional
experiments to examine the status of p27Kip1 in NIH3T3
cells shortly after PAX3-FKHR expression was induced by retroviral
infection. Proliferating NIH3T3 cells were infected with retroviral
stock at increasing multiplicities of infection for 12 h and
harvested at 12 h post-infection for Western blot analysis. As
shown in Fig. 2B, we observed that p27Kip1
protein was specifically decreased in response to increasing amounts of
PAX3-FKHR expression. Furthermore, the decrease in p27Kip1
protein expression was specific for the oncogenic form of Pax3 because
Pax3-expressing cells had a similar level of p27Kip1 as
control cells. This result suggested that suppression of
p27Kip1 protein expression was likely to play a critical
role in the oncogenic function of PAX3-FKHR fusion protein.
In normal cycling cells, the level of p27Kip1 protein
accumulates to maximum levels during G0/G1 and
declines rapidly in late G1 before cells enter S phase. To
determine whether PAX3-FKHR expression affected the rate of
p27Kip1 protein disappearance during the transition from
G0/G1 to S phase, we examined the
p27Kip1 protein profile in the control and
PAX3-FKHR-transformed cells under synchronized conditions. The
PAX3-FKHR-transformed cells required a longer time to exit the cell
cycle than the control cells upon serum withdrawal. As shown by the
fluorescence-activated cell sorter analysis in Fig.
3A, it took close to 60 h
of serum deprivation (0.5% serum) for PAX3-FKHR-transformed cells to
achieve G0/G1 arrest, whereas it took 24 h
to achieve a similar status for the control cells. Control and
PAX3-FKHR-transformed cells were synchronized to
G0/G1 by 60 h of serum deprivation (0.5% serum), then they were stimulated to re-enter the cell cycle with the
addition of growth medium (10% serum). At the indicated time points,
cells were collected and split into two pools. One pool was used to
determine p27Kip1 protein level, and the other pool was
used to verify the functional consequences of the loss of
p27Kip1 protein expression by measuring the activities of
cyclin E and cyclin A-associated Cdk2 complexes that constitute the
major targets of p27Kip1 inhibitory effect (35, 61). As
shown in Fig. 3B, serum stimulation of PAX3-FKHR cells
resulted in a much more rapid decrease in p27Kip1 protein
than in the control cells. Consistent with the p27Kip1
down-regulation, the PAX3-FKHR-expressing cells displayed early activation of both cyclin E- and cyclin A-associated kinase
activities.
The Expression of p27Kip1 in PAX3-FKHR-transformed
Cells Is Controlled at the Post-transcriptional Level--
The change
in p27Kip1 protein levels in normal cells can be regulated
at multiple levels including transcription (44, 45), translation
(46-48), and post-translational modifications (49-52). Because
PAX3-FKHR acts as a transcription factor, we examined whether the
change in p27Kip1 protein expression induced by PAX3-FKHR
was reflected by changes in p27Kip1 mRNA content. As
shown in Fig. 4, the Northern blot
analysis revealed that the level of p27Kip1 mRNA was
unaffected by PAX3-FKHR expression (Fig. 4A), whereas the
level of p27Kip1 protein was (Fig. 4B). We next
examined whether the reduced p27Kip1 protein level was due
to changes in protein stability by carrying out cycloheximide treatment
(Fig. 5A) and in
vivo pulse-chase labeling (Fig. 5B) experiments to
compare the stability of p27Kip1 protein between vector and
PAX3-FKHR expressing NIH3T3 cells. Results from both experiments showed
that the half-life of p27Kip1 protein was dramatically
shortened in PAX3-FKHR-expressing cells. Collectively, these data
suggested that altered regulation of p27Kip1 protein
stability, not changes in transcription, translation, or mRNA
stability, was a major cause in the reduced level of
p27Kip1 protein expression in PAX3-FKHR-transformed
cells.
PAX3-FKHR Promotes 26 S Proteasome-dependent
Degradation of p27Kip1 Protein--
Previous studies show
that ubiquitination-mediated degradation of p27Kip1 protein
by 26 S proteasome plays an important role in regulating p27Kip1 protein level during normal cell cycle progression
from G0/G1 to S phase (50, 62-66). Because
PAX3-FKHR-transformed cells exhibited an accelerated
G0/G1 to S transition (Fig. 3) accompanied by
an increased destabilization in p27Kip1 protein (Fig. 5),
we hypothesized that the p27Kip1 degradation process by the
26 S proteasome system was disrupted in PAX3-FKHR-transformed cells. To
test this, we used protease inhibitors such as MG132, lactacystin, and
high concentrations of
N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal,
which are known to block the 26 S proteasome activity to see if the
p27Kip1 protein level could be restored to normal in
PAX3-FKHR-transformed cells. As shown in Fig.
6A, all three inhibitors were
able to induce dramatic increase in the p27Kip1 protein
level in PAX3-FKHR-transformed cells. In particular, both MG132 and
lactacystin inhibitors were able to bring the p27Kip1
protein level close to the level observed in control cells. In contrast, non-26 S proteasome inhibitors such as caspases inhibitors were ineffective in raising the p27Kip1 protein level in
PAX3-FKHR-expressing cells (Fig. 6A, last
lane).
p27Kip1 is a substrate of cyclin E-Cdk2 and cyclin A-Cdk2
kinase complexes in vivo and in vitro,
respectively, and the phosphorylation of p27Kip1 by Cdk2
kinase is mainly at threonine 187 residue (50, 67, 68). It has been
shown that Cdk2-mediated phosphorylation of p27Kip1 protein
at Thr187 in vivo is required for ubiquitination
and subsequent degradation of p27Kip1 protein in the late
G1 phase of cell cycle (65). As shown in Fig.
3B, PAX3-FKHR-expressing cells rapidly increased both cyclin E- and cyclin A-associated Cdk2 activities after release from serum
starvation. To examine whether Cdk2-dependent
phosphorylation of p27Kip1 protein was critical to the
PAX3-FKHR-induced effect on p27Kip1 protein in
vivo, we utilized chemical inhibitor roscovitine to block Cdk2
kinase activity. Proliferating cells were treated with roscovitine for
12 h, and the level of p27Kip1 expression was
determined by Western blot analysis. As shown in Fig. 6B,
roscovitine treatment of PAX3-FKHR-transformed cells restored
p27Kip1 protein to levels similar to the levels observed in
control cells, suggesting that phosphorylation of p27Kip1
by Cdk2 kinase was involved PAX3-FKHR-targeted degradation of the
p27Kip1 protein.
To verify that phosphorylation of p27Kip1 on
Thr187 residue was critical for the decreased stability of
the p27Kip1 protein by PAX3-FKHR, we developed an in
vitro degradation assay. In this assay,
S35-radiolabled in vitro translated
p27Kip1 protein substrate was incubated with cytoplasmic
extracts prepared from either control or PAX3-FKHR-transformed cells
for 1 h at 30 °C. Under this reaction condition, we were able
to show that the wild type p27Kip1 substrate was more
rapidly degraded by PAX3-FKHR cell extract than control cell extract
(Fig. 7A). This enhanced
degradation activity in PAX3-FKHR cell extract appeared to specifically
involve the 26 S proteasome pathway since the activity was blocked when the non-hydrolyzable form of ATP (ATP
Recent studies show that phosphorylation of p27Kip1 protein
on Thr187 provides a binding site for SCF (Skp1-Cullin-F
box protein), an E3 ubiquitin ligase complex that is responsible for
p27Kip1 polyubiquitination and its subsequent degradation
by the 26 S proteasome pathway (57, 63). The F-box protein is the
substrate-specific recognition component of the SCF complex that is
used to target specific proteins for degradation. In the case of the
p27Kip1 protein, this substrate-specific F-box protein is
Skp2 (58, 66). We investigated whether PAX3-FKHR altered the expression of one or more subunits within the SCF complex that might lead to
increased ubiquitination and subsequent degradation of
p27Kip1 protein. As shown in Fig.
8A, although the protein
levels of Skp1 and CulI remained unchanged between control and
PAX3-FKHR-transformed cells, the level of Skp2 protein was
significantly increased in PAX3-FKHR-transformed cells. Furthermore, we
showed that increased expression of PAX3-FKHR achieved by varying the
multiplicity of retroviral infection led to increased Skp2 expression
that appeared to correlate with decreased levels of
p27Kip1. This effect was not observed in control cells or
in cells expressing increasing levels of Pax3 protein (Fig.
8B). Unlike p27Kip1 gene, the half-life of Skp2
was unchanged in PAX3-FKHR-expressing NIH3T3 cells (data not shown).
Instead, the induction of Skp2 protein level by PAX3-FKHR was
correlated with an increased accumulation of Skp2 mRNA (Fig.
8C). The altered expression of Skp2 and p27Kip1
levels was similarly observed in cells derived from aRMS tumors containing the t(2;13) chromosomal translocation (Fig.
8D).
Exogenous Expression of the p27Kip1 Gene Impairs the
Hyperproliferative and Colony-forming Activities in
PAX3-FKHR- expressing NIH3T3 Cells--
Finally, we addressed the
question of whether overexpression of p27Kip1 protein could
affect the growth rate and colony formation of PAX3-FKHR-transformed
cells. To this end, PAX3-FKHR-transformed cells were stably transfected
with pcDNA3 encoding the mouse p27Kip1 gene or not.
Transfection of the empty vector did not affect the p27Kip1
protein expression levels in PAX3-FKHR-transformed cells (third lane, Fig. 9A). We
screened several p27Kip1 transfectant PAX3-FKHR-expressing
clones to identify ones (an example of one such clone is shown in the
second lane, Fig. 9A) that expressed
p27Kip1 protein at a level comparable with that observed in
untransformed NIH3T3 cells (first lane, Fig. 9A).
We showed that increasing the p27Kip1 protein levels in
PAX3-FKHR-transformed cells significantly slowed their growth rate
(Fig. 9B) and drastically inhibited their contact inhibition-independent colony-forming activity (Fig. 9C).
The biological assays were carried out in three independently isolated p27Kip1 overexpressing stable cell lines with similar
results. These data suggested that suppression of p27Kip1
protein expression by PAX3-FKHR played an active role in oncogenic activity of the fusion protein.
Rhabdomyosarcoma is a soft tissue sarcoma of children and young
adolescents. The most prevalent subtypes of RMS are the embryonal RMS
and the alveolar RMS, with the latter closely associated with higher
disease stage and a poor prognostic outcome (4-8). Unlike embryonal
RMS tumors that do not contain consistent chromosomal aberrations, more
than 90% of aRMS tumors contain a unique t(2,13)(q35;q14) chromosomal
translocation. The molecular consequence of such translocation is the
formation of a novel chimeric protein PAX3-FKHR. Several lines of
evidence strongly suggest that the PAX3-FKHR fusion protein plays an
active role in RMS tumor development. Ectopic expression of PAX3-FKHR
induces accelerated cell proliferation in embryonic fibroblast cells
(31-33, 56) and murine C2C12 myogenic
cells2 and blocks terminal
differentiation in myoblast cells (59). An antisense-induced block of
PAX3-FKHR activity in rhabdomyosarcoma cells is shown to significantly
impair the growth of these tumor cells (55). Clinically, aRMS tumors
are a more aggressive subtype, often showing invasive metastatic
behavior than embryonal tumors. This difference in clinical behavior
may be explained by the presence of PAX3-FKHR fusion protein in the
alveolar subtype. Anderson et al. (60) recently showed that
ectopic expression of PAX3-FKHR into the embryonal RMS cell line
increases cell proliferation and promotes the embryonal RMS cells to
form faster growing, more invasive tumors (60). Taken together, these
studies strongly support the notion that PAX3-FKHR fusion protein has a
pleiotropic and dominant effect on tumor development. However, despite
the overwhelming evidence supporting a dominant oncogenic role for PAX3-FKHR, little is known about the mechanisms involved in altering the cell cycle pathways that lead to uncontrolled growth phenotype.
In this report, we have presented evidence to demonstrate that
PAX3-FKHR-transformed cells proliferate much faster than control and
Pax3-expressing cells. The PAX3-FKHR-expressing cells are more
resistant to serum starvation-induced cell cycle arrest and are able to
enter S phase much more quickly upon serum stimulation. At a molecular
level, we have shown that ectopic PAX3-FKHR expression drastically
suppresses the steady state level of p27Kip1 protein in
exponentially growing NIH3T3 cells. The p27Kip1 protein is
a member of the CIP-KIP family of Cdk2 inhibitors. It has been shown
that the timed decrease in the level of p27Kip1 protein in
late G1 is crucial in promoting S-phase cell cycle transition from G1. Here, we demonstrate that upon
serum-induced cell cycle re-entry, the rate of decline of
p27Kip1 protein is drastically increased in
PAX3-FKHR-transformed cells as compared with control cells. We propose
that by actively reducing the level of p27Kip1 protein in
cells, PAX3-FKHR is able to effectively alleviate the inhibitory effect
on its two major targets, the cyclin A/Cdk2 and cyclin E/Cdk2
complexes. As such, the increased cyclin/Cdk2 activities mediate and
sustain the hyperphosphorylated form of Rb that is required to release
its association with E2F complexes and allow transcription of several
E2F-responsive genes essential for DNA synthesis and for early
commitment of cells to S phase. This idea is supported by the findings
that PAX3-FKHR elevates the overall level of hyperphosphorylated form
of Rb.
In addition to shortening of G1 phase, we have also
observed that the PAX3-FKHR-transformed cells are less sensitive to
serum starvation than the control cells in that they require nearly 60 h of low serum (0.5% serum) treatment to be properly arrested at G0/G1, whereas the control cells arrest
within 24 h of serum starvation. These data suggest that ectopic
expression of PAX3-FKHR does not induce mitogen independence in NIH3T3
cells but rather significantly delays the cell response to negative
growth signal. Because PAX3-FKHR-expressing cells are able to
accumulate p27Kip1 protein to the same level as in control
cells upon prolonged serum deprivation, it is unlikely that
PAX3-FKHR-transformed cells have a defective mechanism in
p27Kip1 protein synthesis; rather, the transformed cells
require a longer time to increase p27Kip1 levels due to the
robust degradation of this protein. It is likely that additional
mechanisms are also involved in delayed growth arrest response in
PAX3-FKHR-transformed cells. For example, a recent study has shown that
PAX3-FKHR can directly transcriptionally activate the promoter of IGF
receptor gene (69). If so, the higher density of IGF receptor in these
cells might be sufficient enough to carry cells through extra rounds of
cell cycle at low serum condition.
Although the level of p27Kip1 mRNA stays relatively
unchanged throughout cell cycle in normal cells, suppression of
p27Kip1 transcription has been reported in cells expressing
viral oncoprotein v-src (44). Moreover, we become intrigued by the
possibility that PAX3-FKHR might affect p27Kip1 expression
through altered gene transcription because 1) PAX3-FKHR normally acts
as a transcription factor, 2) we have shown in previous study that
PAX3-FKHR fusion protein has a gain of function in that it can alter
expression of genes that are not normally targeted by Pax3 (27), and 3)
the normal FKHR counterpart of the fusion protein has been shown to
play a role in transcriptional regulation of p27Kip1 after
mitogen induction (45). However, we are unable to detect a change in
the total amount of p27Kip1 mRNA in
PAX3-FKHR-transformed cells, suggesting p27Kip1 is not a
direct target for transcription activation by PAX3-FKHR. Instead, we
find that the predominant effect of PAX3-FKHR on p27Kip1 is
through an enhanced degradation of p27Kip1 protein. We have
provided several lines of evidence to support this conclusion. First,
experiments using the protein synthesis inhibitor cycloheximide as well
as in vivo pulse-chase labeling studies show that the
stability of p27Kip1 protein is dramatically reduced in
PAX3-FKHR-expressing cells. Second, we show that protease inhibitors
specific for 26 S proteasome activity prevent degradation of
p27Kip1 protein and restore p27Kip1 protein
levels in PAX3-FKHR cells to the levels found in control cells. Third,
we show that cytoplasmic protein extract prepared from
PAX3-FKHR-transformed cells can degrade in vitro translated wild type p27Kip1 substrate much more efficiently than
extracts prepared from the control cells. Similar to the in
vivo result, 26 S proteasome inhibitors specifically block the
in vitro degradation activity. Collectively, these results
suggest that p27Kip1 stability is decreased by PAX3-FKHR
through increased ubiquitin-dependent degradation by the 26 S proteasome pathway.
Down-regulation of p27Kip1 protein expression by PAX3-FKHR
appears to be a relatively early event since this effect is observed within 12 h after PAX3-FKHR protein is induced in the cells (Fig. 3B). Because PAX3-FKHR is a transcription factor, it seems
unlikely that PAX3-FKHR is directly involved in the timed increase in
the degradation of p27Kip1 protein. In the in
vitro degradation assay, we have determined that the addition of
PAX3-FKHR protein to control cell extract is not sufficient to cause
accelerated p27Kip1 protein degradation (data not shown).
An alternative explanation would be that additional cellular components
that are specifically activated in PAX3-FKHR-transformed cells are
required to induce p27Kip1 protein degradation. As
mentioned earlier, IGF receptor has been shown to be a direct target
for transcription activation by PAX3-FKHR. Increased IGF receptor
expression would lead to an activation of mitogen-induced signal
transduction pathways that may alter the p27Kip1 level.
However, in our PAX3-FKHR-transformed NIH3T3 cells, we were unable to
detect an effect on p27Kip1 protein levels by chemical
blockers that inhibit Akt/protein kinase B, extracellular
signal-regulated kinase/mitogen-activated protein kinase (MAPK), p38
MAPK, and p70S6 kinase mediated activities (data not shown).
In this report, we have found that the expression of F-box protein
Skp2, the substrate-specific E3 ubiquitin ligase of SCF complex
(Skp1/Cul1/F-box), is selectively up-regulated in PAX3-FKHR-transformed cells. Both biochemical and genetic experiments have implicated that
Skp2 is responsible for the ubiquitin-dependent degradation of p27Kip1 protein in vivo and in
vitro. Binding of Skp2 to p27Kip1 protein requires
phosphorylation of p27Kip1 on Thr-187 by cyclin-Cdk2
complexes (50, 65, 66). In our study, we have shown that inhibition of
Cdk2 activity by roscovitine increases p27Kip1 protein
levels in PAX3-FKHR-transformed cells in vivo (Fig.
7B) and the p27Kip1 with mutation to the Thr-187
residue is resistant to degradation by extracts from PAX3-FKHR cells
in vitro (Fig. 8B). Presumably, elevation of Skp2
protein by PAX3-FKHR would permit accelerated ubiquitination of
p27Kip1, which is a prerequisite step for targeted
degradation of p27Kip1 protein by the 26 S proteasome
pathway. This notion is in line with the observation of a PAX3-FKHR
dose-dependent, inverse correlation between the expression
of Skp2 and p27Kip1 proteins. The finding that Skp2 RNA
content is increased in the PAX3-FKHR-expressing NIH3T3 and aRMS tumor
cells leads us to hypothesize that the direct target for PAX3-FKHR
effect on p27Kip1 may be Skp2 transcription. It will be
important in the future to test this idea by determining whether
PAX3-FKHR can directly transactivate Skp2 promoter. Furthermore, the
activation of Skp2 gene expression is likely to affect degradation of
other proteins, and it will be important to identify additional targets
and examine their role in PAX3-FKHR oncogenesis.
In summary, the present study has presented clear evidence to show that
the PAX3-FKHR selectively accelerates p27Kip1 protein
degradation by the ubiquitin-dependent 26 S proteasome pathway. The targeted destabilization of p27Kip1 protein by
PAX3-FKHR serves as an active mechanism in PAX3-FKHR transformation
since restoration of p27Kip1 level through ectopic
expression greatly reduces the hyperproliferation and contact
inhibition-independent growth properties of PAX3-FKHR-transformed cells. Moreover, we have identified Skp2 as a potential regulatory link
between PAX3-FKHR activation and p27Kip1 down-regulation.
We have used NIH3T3 cells as our model system because it offers the
opportunity to identify proximal targets whose expression/function is
changed as a consequence of PAX3-FKHR expression, whereas in tumor
cells it is likely that there have been extensive genetic mutations
acquired during the progression from transformation to tumor. In this
report, we are able to verify that both p27Kip1 and Skp2
are similarly dysregulated in two PAX3-FKHR-expressing aRMS cell lines
as compared with normal skeletal muscle. In future work, it will be
important to determine whether these alterations are common to all RMS
or more specific to a particular RMS subtype. It is possible that the
fusion process induces a conformational change within PAX3-FKHR,
enabling it to regulate the expression and function of genes that are
not regulated by its normal counterparts under normal physiological
conditions. Thus, identification of genes whose expression and function
are selectively disrupted by the oncogenic form of Pax3 is essential to
gain a full understanding of the mechanistic links between PAX3-FKHR
and oncogenic transformation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-tubulin (ab-1) was from Oncogene. In vitro
translated p27Kip1 protein was generated by the use of the
T7-coupled TNT in vitro transcription-translation kit under
conditions recommended by the manufacturer (Promega). Total RNA and
protein extract from human skeletal muscle were purchased from Stratagene.
80 °C
until use. Cytoplasmic cell extract was prepared from cells by first
swelling in low salt hypotonic buffer (20 mM HEPES, pH 7.5, 1.5 mM MgCl2, 5 mM KCl, 1 mM DTT) for 30-60 min on ice before lysis with a hand-held Dounce homogenizer. Preparation was examined under light microscopy to
ensure the nuclei remained intact during homogenization. The cytoplasmic extract was cleared by centrifugation at 10,000 × g
for 30 min at 4 °C and stored at
80 °C until use.
80 °C.
-glycerophosphate) before
setting up the kinase reaction. The cyclin A- and cyclin E-associated
Cdk2 kinase reactions were performed by resuspending the washed
immunocomplex beads in a final volume of 20 µl of kinase buffer
containing 20 mM ATP, 0.1 mM protein kinase A
inhibitor, 10 µCi of [
-32P]ATP, and 1 µg of
histone H1. The reaction was carried out for 30 min at 30 °C,
terminated by the addition of Laemmli sample buffer, and analyzed on a
10% SDS-polyacrylamide gel.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
PAX3-FKHR induces hyperproliferation in
NIH3T3 fibroblast cells. NIH3T3 cell lines that ectopically
expressed vector (open circles), PAX3-FKHR
(filled squares), and Pax3 (crosses)
gene were seed at 1 × 104 cells per well in 12-well
plates at day 0. At a six-day experimental period, cells were harvested
each day into phosphate-buffered saline and stained with trypan blue.
Only viable cells were counted. The experiment was carried out more
than three times on different days using different lots of pooled cell
lines.
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Fig. 2.
Western blot analysis of cell cycle
regulatory proteins by PAX3-FKHR in NIH3T3 cells. A, a
total of 25 µg of high salt extract prepared from proliferating
control or PAX3-FKHR-transformed (PF) NIH3T3 cells was
size-fractionated by SDS-PAGE followed by immunodetection with
antibodies specific for different cell cycle regulatory proteins.
p16 was analyzed in primary mouse embryonic fibroblast cells.
B, PAX3-FKHR down-regulates p27Kip1
protein expression in NIH3T3 cells in a dose-dependent
manner. Proliferating NIH3T3 cells (2 × 105
cells/60-mm dish) were infected with retrovirus-expressing vector,
Pax3, or PAX3-FKHR at increasing multiplicities of infection
(M.O.I.) overnight. Cells were collected for 12 h
post-infection for high salt extract preparation. A total of 25 µg of
extract was examined by SDS-PAGE followed by antibody detection.
-Tubulin antibody was used for sample normalization.
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Fig. 3.
Analysis of the effect of PAX3-FKHR on the
expression of p27Kip1 protein and activities of cyclin
E-Cdk2 and cyclin A-Cdk2 kinases in synchronous culture.
A, fluorescence-activated cell sorter analysis of growing
and synchronized control and PAX3-FKHR-transformed cells. B,
duplicate plates of growth-arrested (60 h) serum starvation control or
PAX3-FKHR-transformed NIH3T3 cells were stimulated with 10% bovine
calf serum containing media to enter cell cycle. At the
indicated times after serum stimulation, one set of cells was extracted
with high salt buffer, and a total of 30 µg of extract was used to
detect the expression of PAX3-FKHR and p27Kip1. The second
set of cells was extracted with immunoprecipitation buffer, and a total
of 200 µg of cell extract was used in the immunoprecipitation
reaction in the presence of either cyclin A or cyclin E antibody
followed by the kinase reaction as described under "Materials and
Methods."
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Fig. 4.
Decreased p27Kip1 expression in
PAX3-FKHR expressing NIH3T3 cells is not due to changes in
p27Kip1 mRNA content. Duplicate plates of NIH3T3
cells expressing either vector, PAX3-FKHR, or Pax3 with or without
expression of Pax3 and PAX3-FKHR were used to prepare total RNA for
Northern blot analysis (A) and Western blot analysis
(B), respectively. For Northern blot analysis, a total of 15 µg was used to determine p27Kip1 mRNA content. For
sample normalization, the blot was stripped and re-probed with
32P-labeled DNA corresponding to the rat
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sequence.
The membrane was scanned by phosphorimaging for quantitative analysis.
The signal (corrected for glyceraldehyde-3-phosphate dehydrogenase)
detected in control cells that were transfected with empty vector was
assigned an arbitrary value of 1. Fold induction is the ratio of the
signal in PAX3-FKHR-expressing cells to that of control cells. For
Western blot analysis, a total of 25 µg of radioimmune precipitation
assay buffer extract was used to detect the expression of Pax3,
PAX3-FKHR, and p27Kip1 proteins.
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Fig. 5.
PAX3-FKHR induces destabilization of
p27Kip1 protein. A, measurement of
p27Kip1 protein degradation after inhibition of protein
synthesis by cycloheximide (CHX). Proliferating NIH3T3 cells
(2 × 105 cells/60-mm dish) were infected with the
vector or PAX3-FKHR-expressing retrovirus (PF). Twenty-four
hours after infection, cells were treated with 10 µg/ml cycloheximide
for the indicated times, and p27Kip1 protein levels were
followed by Western blot analysis. B, measurement of
p27Kip1 protein stability by an in vivo
pulse-chase labeling method. Proliferating control and
PAX3-FKHR-transformed NIH3T3 cell lines were pulse-labeled in media
containing [35S]Met and later chased with media
containing unlabeled Met for the indicated times as described under
"Materials and Methods." Radiolabeled p27Kip1 and
-tubulin from the radioimmune precipitation assay buffer cell
extracts were immunoprecipitated with anti-p27Kip1 and
anti-
-tubulin antibodies, and the immunocomplexes were pulled down
with protein G-Sepharose beads. The immunocomplexes were separated by
SDS-PAGE, and radioactive protein products were visualized by
autoradiography.
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Fig. 6.
Inhibition of 26 S proteasome
(A) or Cdk2 (B) activities increases
p27Kip1 levels in PAX3-FKHR-transformed cells.
Proliferating control and PAX3-FKHR-transformed (PF) NIH3T3
cells were treated with Me2SO (DMSO) solvent or
indicated inhibitors for 6 h (A) or 12 h
(B) before harvest. High salt extracts (25 µg) of each
sample were analyzed by Western blot. Inhibitors used in A
were 26 S proteasome inhibitors (10 µM MG132, 10 µM lactacystin, 100 µM
N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal
(ALLN)), and caspase inhibitor (Inh.) III mixture
(50 µM).
S) was used in place of the
ATP-regenerating system (Fig. 7B), and the activity was
specifically blocked in the presence of 26 S proteasome inhibitors
(Fig. 7C). Moreover, we showed that
Cdk2-dependent phosphorylation of p27Kip1 on
Thr187 residue was required for the PAX3-FKHR effect
because mutation of Thr187 to Ala in the
p27Kip1 substrate completely prevented its degradation by
PAX3-FKHR cell extract (Fig. 7D). Collectively, the in
vivo (Fig. 6) and in vitro (Fig. 7) analyses strongly
suggested that PAX3-FKHR promoted destabilization of
p27Kip1 protein in cells by altering the activity of
ubiquitin-dependent 26 S proteasome degradation
pathway.
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Fig. 7.
Cytoplasmic extract of PAX3-FKHR expressing
cells contains highly active proteolytic activity toward
p27Kip1 protein in an in vitro cell-free
degradation system. A total of 25 µg of hypotonic extract
prepared from proliferating control and PAX3-FKHR-transformed
(PF) NIH3T3 cells was incubated with 0.3 fmol of
35S-labeled in vitro translated wild type
p27Kip1 for the indicated times (A) or
35S-labeled in vitro translated T187A mutant
p27Kip1 for 1 h (D). The reaction was
terminated by the addition of SDS sample buffer and resolved by
SDS-PAGE followed by autoradiography. The effect of PAX3-FKHR on
p27Kip1 degradation was tested for dependence on ATP by
replacing the ATP-regenerating system with 5 mM
non-hydrolyzable ATP S (B) and on 26 S proteasome activity by
carrying out the degradation reaction in the presence of
Me2SO (DMSO), MG132, lactacystin, and caspases
inhibitor mixture (C). RRL, rabbit reticulocyte
lysate.
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Fig. 8.
Expression of Skp2 is up-regulated in
PAX3-FKHR-expressing NIH3T3 cells (A-C) and in
PAX3-FKHR-expressing rhabdomyosarcoma cells
(D). For analyzing protein expression levels, a
total of 25 µg of radioimmune precipitation assay buffer extract,
prepared from control and PAX3-FKHR-transformed NIH3T3 cell lines
(A), NIH3T3 cells (2 × 105 cells/60-mm
dish) that were transiently infected with retrovirus-expressing vector,
Pax3, or PAX3-FKHR at increasing multiplicities of infection
(M.O.I., B), or normal skeletal muscle and
PAX3-FKHR-expressing alveolar rhabdomyosarcoma cells (D,
left panel) was used for Western blot procedure. For
analyzing mRNA expression level, a total of 15 µg of RNA was
prepared from NIH3T3 cells that were infected with
retrovirus-expressing vector or PAX3-FKHR (C). In
D, protein (left panel) and RNA (right
panel) isolated from normal skeletal muscle (Sk. musc.)
and PAX3-FKHR-expressing alveolar rhabdomyosarcoma cells was analyzed
for Skp2 and p27Kip1 expression. For normalization, the
membrane was stripped and re-probed either with radiolabeled
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA
probe (C) or 36B4 DNA probe (D, left
panel). -Tubulin antibody was used to normalize sample
loading.
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Fig. 9.
Ectopic expression of p27Kip1
blocks oncogenic activity in PAX3-FKHR-transformed NIH3T3 cells.
PAX3-FKHR-transformed cells were established by stable selection
(puromycin) of NIH3T3 cells infected with pBabe-PAX3-FKHR-expressing
retrovirus. Control cells were established from NIH3T3 cells infected
with pBabe-expressing retrovirus. These PAX3-FKHR-transformed cells
were transfected with a total 20 µg of DNA containing either
pcDNA3 vector or pcDNA3 vector expressing p27Kip1
cDNA by CaPO4 precipitation method. Stably transfected
cells were selected in media containing 400 µg/ml G418 for 21 days,
and individual clones were isolated, analyzed for expression of
p27Kip1, and expanded for experimentation. A,
the expression levels of PAX3-FKHR and p27Kip1 proteins
were assessed by Western blot analysis. Protein extract prepared from
untransfected control cells (pBabe-expressing) was included in the
analysis to establish the base-line expression level of
p27Kip1 in non-transformed NIH3T3 cells. B, cell
proliferation assay. PAX3-FKHR-expressing stable cell line transfected
with expressed pcDNA3 vector or p27Kip1 expression
vector was seeded at 2 × 104 cells/well in 12-well
plates. At the indicated times, cells were rinsed twice with
phosphate-buffered saline, trypsinized, and counted under the same
conditions as described in the Fig. 1 legend. C, colony
formation assay. Confluent PAX3-FKHR-expressing stable cells that
expressed pcDNA3 vector or p27Kip1 were cultured in a
monolayer for an additional 10 days to allow colonies to form. Cells
were replenished with fresh media every other day. For visualization,
cells were rinsed twice with phosphate-buffered saline, fixed in
formalin solution, and stained with 2% crystal blue solution.
Background staining was removed by repeated rinsing with 10% ethanol
solution before photography.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Hiroaki Kiyokawa for his helpful suggestions in this study. We also thank Dr. Reed Graves for help in proofreading of this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant CA-74907 (to C. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: CMBOD, University of
Illinois at Chicago, 801South Paulina St., Chicago, IL 60612. Tel.:
312-996-4530; Fax: 312-413-1604; E-mail:
chiayeng@tigger.cc.uic.edu.
Published, JBC Papers in Press, October 24, 2002, DOI 10.1074/jbc.M205424200
2 C. Wang, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
aRMS, alveolar
rhabdomyosarcoma;
DTT, dithiothreitol;
IGF, insulin-like growth factor;
ATPS, adenosine 5'-O-(thiotriphosphate).
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REFERENCES |
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