From the Department of Microbiology and Immunology
and Cellular and Molecular Biology Graduate Program, University of
Michigan Medical School, Ann Arbor, Michigan 48109-0620 and the
¶ Comprehensive Cancer Center, University of Michigan Medical
Center, University of Michigan, Ann Arbor, Michigan 48109
Received for publication, September 19, 2000, and in revised form, October 16, 2000
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ABSTRACT |
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Prothymosin Prothymosin ProT c-myc is a proto-oncogene that has been implicated in normal
proliferation and diverse forms of tumorigenesis (21). Overexpression of c-myc stimulates cell cycle progression, and induces cell
transformation and apoptosis (22). The transcriptional activation of
c-myc leads to an increase in the level of transcription of
ProT Cell Culture and Transfections--
Rat-1 cells, obtained from
George Mosialos and Elliott Kieff, were grown in Dulbecco's modified
Eagle's medium (DMEM) containing 10% fetal bovine serum (Gemini
Bio-products, Inc.), penicillin (25 units/ml), streptomycin (25 µg/ml), and gentamicin (10 µg/ml) and maintained in a humidified
atmosphere at 37 °C with 5% CO2. After two passages,
Rat-1 cells were seeded at 1 × 105 cells/35-mm
diameter six-well plate, and transfected when ~60% confluent with 5 µg of pA3M vector, pA3M-ProT
For studies of serum dependence, 1 × 105 cells were
plated in 35-mm six-well plates containing DMEM with 10% serum. The
cultures were washed twice with serum-free medium and then resuspended in DMEM with 1.0%, 2.5%, 5.0%, and 10% fetal bovine serum. Cells were trypsinized on the fourth day and were counted using a
hemocytometer with trypan blue staining. Photographs were taken at 10×
magnification using a BK40 Olympus phase contrast microscope.
Soft Agar Assay--
Soft agar plates were prepared as described
previously (32). Briefly, the bottom layer was made by melting 1.4%
DNA grade agarose (American Bioanalytical) in sterile water, which was
cooled to 40 °C in a water bath and added to an equal quantity of
1× DMEM enriched with 20% glutamine, and 1.6 mg/ml G418, yielding a
final concentration of 0.7% agar in DMEM. The top agar layer was
prepared by melting 0.7% agar, followed by equilibration at 40 °C
in a water bath. Trypsinized cells (1.8 × 105) grown
under selection for 48 h were added to 5 ml of the 0.7% top agar
and poured onto the prepoured bottom layer in a 100-mm plate. The soft
agar was covered with 1 ml of DMEM containing 10% serum and 800 µg/ml G418 every 5 days and incubated at 37 °C with 5%
CO2. Colonies were observed and quantified 4 weeks after transfection.
Cell Growth Experiment--
To measure the growth of cells,
1 × 105 cells were plated into 35-mm plates and
allowed to grow for 12 days. The cells were trypsinized at 2-day
intervals up to 12 days and counted on a hemocytometer with trypan blue
staining. A total of six time points were taken for each transfection.
This assay was repeated three times, and data points were plotted as
mean ± S.D.
Plasmid Constructs--
ProT Western Blot Analysis--
Rat-1 cells were transfected as
described above with pA3M vector, pA3M-Ras, and pA3M-ProT Induction of Cell Proliferation by ProT ProT ProT ProT The Transformed Rat-1 Foci Express ProT Although it has been implicated in cell proliferation (1-4, 8)
and chromatin remodeling (29, 30), the physiological function of the
ProT The experiments presented here show that ProT The mechanism by which ProT ProT As already stated, ProT Current studies also suggest that the proliferation and transformation
induced by the DNA tumor virus EBV in various human malignancies may in
part involve targeting of ProT Studies have also demonstrated that ProT It is noteworthy that ProT (ProT
), a cellular molecule
known to be associated with cell proliferation, is transcriptionally
up-regulated on expression of c-myc and interacts
with histones in vitro and associates with histone H1 in
cells. Previous studies have also shown that ProT
is involved in
chromatin remodeling. Recent studies have shown that ProT
interacts
with the acetyl transferase p300 and an essential Epstein-Barr virus
protein, EBNA3C, involved in regulation of viral and cellular
transcription. These studies suggest a potential involvement in
regulation of histone acetylation through the association with these
cellular and viral factors. In the current studies, we show that
heterologous expression of ProT
in the Rat-1 rodent fibroblast cell
line results in increased proliferation, loss of contact inhibition,
anchorage-independent growth, and decreased serum dependence. These
phenotypic changes seen in transfected Rat-1 cells are similar to those
observed with a known oncoprotein, Ras, expressed under the control of a heterologous promoter and are characteristic oncogenic growth properties. These results demonstrate that the ProT
gene may function as an oncogene when stably expressed in Rat-1 cells and may be
an important downstream cellular target for inducers of cellular
transformation, which may include Epstein-Barr virus and
c-myc.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(ProT
)1 is implicated in
the growth of normal cells as well as in the proliferation of mammalian
cells undergoing malignant transformation. However, the physiological
role of this protein in cell proliferation remains to be elucidated.
Nonetheless, expression of the gene is generally correlated with
cellular proliferation and is low in quiescent cells (1-4). This
prompted us to determine whether ProT
has any oncogenic potential
for cellular transformation in a rodent fibroblast assay. The present
study indicates that overexpression of ProT
in Rat-1 cells is
sufficient to induce a transformed phenotype of these cells in
vitro that is similar to the phenotype produced by the
ras oncogene when overexpressed under similar conditions.
is a small (12.5 kDa), highly acidic nuclear protein, which
contains a putative nuclear localization signal at the carboxyl terminus and a small basic domain at the amino terminus (1, 5-9).
Additionally, ProT
is highly conserved and ubiquitously expressed in
a wide variety of cells, tissues, and organisms (1-4, 8), which
suggests that it is required for an essential function of the cell.
Subsequently, numerous research findings have indicated that ProT
may be involved in cell proliferation. This relationship has been
supported by the direct correlation of both ProT
mRNA and
protein levels with levels of proliferation. The transcript is induced
upon growth stimulation of resting lymphocytes (4), thymocytes (10),
NIH 3T3 fibroblasts (4, 11), and hepatocytes during liver regeneration
(10, 12). Moreover, ProT
concentrations were reported to be higher
in tumor samples than in normal breast tissue (13), and were
phosphorylated in stimulated proliferating cells (14). ProT
gene
expression is elevated in normal proliferating tissue (15), but
repressed in quiescent cells (13). Expression of ProT
correlates
with proliferation (16), whereas ProT
antisense oligonucleotides
induce apoptosis in HL-60 cells (17). Additionally, ProT
expression
is also known to occur in proliferating B and T lymphocytes (18).
Overexpression of ProT
has been shown to accelerate proliferation,
and to retard HL-60 promyelocyte differentiation (19). Antisense
oligonucleotides of ProT
mRNA have also been shown to inhibit
cell proliferation in myeloma cells (20).
, and ProT
mRNA levels vary with c-myc
expression during tissue proliferation, viral infection, and heat shock
(23). Overexpression of ProT
is concomitant with that of
c-myc during rat hepatic carcinogenesis (24). An E-box
element localized in the first intron mediates transcriptional
regulation of the gene for ProT
by c-myc (25). Furthermore, the human papilloma virus type 16 E6 oncogene can transactivate the ProT
and c-myc promoters (26, 27),
indicating that ProT
is a transcriptional target of at least two
known oncogenes. Conditional expression of c-myc in human
neuroblastoma cells increases ProT
and accelerates early progression
into S-phase after mitogenic stimulation of quiescent cells (28).
Recent studies have shown that ProT
binds to histones in
vitro (29), thereby suggesting a possible role for this molecule
in chromatin remodeling in mammalian cells (7, 30, 31). Despite the
documented evidence for a role of ProT
in cell proliferation, its
potential role in induction and maintenance of cell transformation has
not been investigated. Since ProT
is closely associated with cell
proliferation and is induced upon expression of the known oncogene
c-myc and the human papillomavirus E6 protein, we decided to
investigate the potential role of ProT
when overexpressed in Rat-1
cells by phenotypic transformation assays. Here we show that
overexpression of ProT
induces the typical transformed phenotype
in vitro as shown by increased proliferation,
anchorage-independent growth, loss of contact inhibition, and decreased
serum dependence of the transfected Rat-1 cells, which suggest that
ProT
may function as a cellular oncogene and is likely one of the
important downstream targets for inducers of transformation like
c-myc.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, or pA3M-Ras. 10 µl of Superfect
reagent (Qiagen) and 5 µg of each plasmid were resuspended in 140 µl of DMEM without serum and incubated for 10 min at room
temperature, followed by addition of 1.5 ml of DMEM (with 10% serum).
The cells were placed under selection 24 h after transfection with
DMEM containing 800 µg/ml G418. The cells were trypsinized after
growing for 48 h under selection and plated in DMEM containing
10% fetal bovine serum, penicillin (25 units/ml), streptomycin (25 µg/ml), and gentamicin (10 µg/ml). Photographs were taken at the
beginning of the fourth week, and colonies were counted.
was cloned by ligation of the
entire coding sequence amplified from pAV1 (33) in frame with the Myc
epitope into the pA3M mammalian expression vector (34). Primers
flanking the coding sequence of ProT
contained EcoRI and
EcoRV restriction sites in the forward and reverse primers,
respectively (forward primer, 5'-GGAATTCCATGTCAGACGCAGCCGTAGACA-3';
reverse primer, 5'-GGATATCGGGTCATCCTCGTCGGTCTTCTG-3') and were used to
amplify ProT
cDNA with Vent polymerase (New England Biolabs).
The ras cDNA was cloned in a similar fashion using
forward and reverse primers (5'-TTAGAATTCATGACAGAATACAAGCTTGTG-3' and
5'-TTAGATATCTAGGACAGCACACACTTGCA-3', respectively). The polymerase
chain reaction product obtained was ligated into the prepared
EcoRI and EcoRV sites of pA3M in frame with the
Myc epitope tag at the carboxyl terminus.
, followed
by several weeks of clonal selection in 800 µg/ml G418 (Life
Technologies, Inc.). Approximately 5 × 105 cells were
lysed in SDS-lysis buffer and fractionated on a 15% SDS-polyacrylamide
gel, followed by electrotransfer to a 0.22-µm nitrocellulose membrane
(Micron Separations, Inc.). Recombinant ProT
was detected with
monoclonal anti-Myc antibodies (clone 9E10, ATCC), followed by
incubation with anti-mouse horseradish peroxidase antibodies (1:2500,
Amersham Pharmacia Biotech) and standard chemiluminescence protocol as
suggested by manufacturer (Amersham Pharmacia Biotech).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
Rat-1 cells
were transfected with ProT
, Ras, or pA3M vector alone. Transfected
cells were grown in DMEM complete medium with 10% fetal bovine serum
and 800 µg/ml neomycin. The cells were trypsinized and counted at
2-day intervals up to 12 days when cell transfected with Ras, and
ProT
reached confluence. Little difference was noted up to 6 days,
during which time the Ras- and ProT
-transfected cultures
proliferated at rates similar to that observed for the control vector
transfectants. After 6 days, however, the rate of proliferation in
cells transfected with ProT
and Ras was substantially greater than
that observed in cells transfected with empty pA3M vector. This trend
continued until day 12, at which time the Ras and ProT
cultures
reached confluency (Fig. 1).
Interestingly, ProT
demonstrated an enhanced ability to induce
proliferation when compared with the Ras-transfected cells (Fig. 1).
This suggests that ProT
may have more potent effects on cell cycle
by driving the cells through the G1 phase more rapidly than
those effects that are due to Ras expression. Each data point is based
on counts from multiple transfections done in triplicate. These results
clearly show that ProT
induces proliferation of the rodent
fibroblast Rat-1 cell line in vitro in a similar manner to
the known oncoprotein Ras, which indicates that these closely related
phenotypes are induced in vitro by these two cellular
proteins.
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Fig. 1.
Growth rate in Rat-1 cell clones. Rat-1
cells were passaged twice and plated in 35-mm six-well plates. The
cells were transfected at 60% confluence with pA3M vector, pA3M-Ras,
and pA3M-ProT . Cells were selected with 800 µg/ml G418 after
24 h. Cells were trypsinized every 2 days from a new well, and
counted on hemocytometer. Cells were counted in duplicate, with three
independent readings. Data points represent mean values. Standard
deviations ranged from 3% to 22%. Triangles indicate
ProT
, squares indicate Ras, and diamonds
indicate vector control. Starting at day 6 and continuing through the
completion of the experiment at day 12, ProT
and Ras Rat-1 clones
displayed a significantly increased growth rate compared with vector
control.
Induces Foci Formation in Monolayer Cultures--
Rat-1
cells were transfected with pA3M-ProT
or pA3M-Ras expression vector
as well as the pA3M vector alone, followed by selection with neomycin
after 24 h. Phenotypic changes were observed 20 days after
transfection for the Ras and ProT
transfections but not in vector
alone control. Little or no foci formation was observed in cultures
that were transfected with vector alone (Fig.
2, panel A and
D) in which the cells under selection continued to grow in a
monolayer until they reached confluency. However, in the Ras- and
ProT
-transfected cultures, many foci were readily apparent by 20 days after transfection. This phenotypic change was macroscopically visible on average between 18-21 days after transfection but could be
seen within 10 days microscopically. The foci in these cultures were
typically 1-2 mm in size and continued to grow in size as they
remained in culture. Foci that developed from Ras-transfected cells
were similar to that of foci developing from ProT
-transfected Rat-1
cells (Fig. 2, compare panels B and E
with panels C and D). The cells in
these foci continued to grow upon one another with an apparent absence
of contact inhibition until they would break off from the dish and
again continue to stack on each other as the foci grew larger. The
number of foci obtained on an average from the Ras-transfected cells
was slightly higher than that seen with ProT
transfected cells
(Table I). However, this was not obvious
by visual inspection when comparing plates containing the transfected
cultures (see Fig. 2, panels B and C).
A small number of foci less than 0.5 mm in size were seen in the vector alone control; however, only foci greater than 1 mm in size were counted. Additionally, the foci formed in the vector alone control were
observed only after the cultures reached confluence and did not grow in
size over time (Fig. 2, panels A and
D), whereas foci were formed in ProT
and Ras cultures at
an earlier time, before the respective monolayer eventually became
confluent. Foci were counted in each of three cultures 25 days after
selection. The data shown in Table I represent three independent
experiments and show that ProT
induces a loss of contact inhibition
phenotype and, like Ras, allows Rat-1 cells to grow upon one another
and form multicellular foci.
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Fig. 2.
ProT induces Rat-1
cells to form foci similar to Ras. Rat-1 cells were passaged twice
and seeded in DMEM containing 10% serum. Cells were transfected with
pA3M vector alone or with vector containing Ras or ProT
cDNA.
Cells were maintained in DMEM containing 10% serum and 800 µg/ml
G418. Representative photomicrographs were taken after 4 weeks of
selection with a phase contrast microscope (magnifications, ×1.5 and
×10) and show the ability of ProT
to induce cellular foci in a
fashion similar to that observed with the known Ras oncoprotein
(compare panels A-C and D-F).
Expression of ProT in Rat-1 fibroblasts induces
anchorage-independent growth similar to that observed by the known
oncogene ras
Promotes Growth of Rat-1 Cells in Low
Serum--
Typically, cells that are transformed have a decreased
dependence on serum for growth. To determine if ProT
is capable of inducing serum-independent growth, cells transfected with Ras, ProT
,
and vector alone were grown in media containing 1.0%, 2.5%, 5.0%,
and 10.0% serum for 4 days. The cells were trypsinized, and viable
cells were counted on a hemocytometer with trypan blue staining.
At low serum concentration (1%), growth was not pronounced for any of
the samples (Fig. 3a,
A-C), whereas at concentrations of 2.5% to 5% vigorous
growth was observed with Rat-1 cells transfected with both Ras or
ProT
, but not in the vector alone control (Fig. 3a,
D-F and G-I). At concentrations of 10% serum,
cell cultures grew at a relatively similar rate. As expected, vector
control cells did not grow significantly in media that contained 1%
serum and showed a clear dose response to serum as the concentration was increased (Fig. 3b). Both ProT
- and Ras-transfected
cultures, however, rapidly reached confluence (Table
II) and similarly achieved much higher
cell density (Fig. 3b) in 2.5%, 5%, and 10% serum concentrations. In all dilutions of serum, 1-10% of the Ras- and ProT
-transfected cells had similar growth properties in terms of
number of cells (Fig. 3b) and morphological properties (Fig. 3a) and levels of confluence (Table II). These results show
that ProT
promotes growth of Rat-1 cells in low serum concentrations with striking similarity to that of the known oncoprotein Ras. This
indicates that cells transfected with ProT
can stimulate cellular
processes required for cell proliferation even under low serum
conditions, again indicating a transformed phenotype.
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Fig. 3.
a, ProT induces serum-independent
growth of Rat-1 cells. After transfection, Rat-1 cells were
grown in 1.0%, 2.5%, 5.0%, and 10.0% serum for 6 days and
photographed with phase contrast microscopy (magnification, ×10) to
show fields that reflect typical cell morphology and density. Rat-1
cells expressing ProT
and Ras display significantly enhanced ability
to grow in low serum concentrations (compare panels
B, E, H, and K and
panels C, F, I, and
L with panels A, D,
G, and J). b, effect of serum on cell
growth. Transfected Rat-1 cells were grown in the presence of 1.0%,
2.5%, 5.0%, and 10.0% serum concentrations for 4 days, at which
time cells were trypsinized and viable cells were counted on a
hemocytometer with trypan blue staining. Data points in Fig.
3b represent mean values ± S.D.
ProT promotes growth of Rat-1 cells in low serum as indicated by
increased levels of confluence compared to vector control
), 25% (+), 50% (++), 75% and confluent
plates denoted by (++++).
Induces Colony Formation of Rat-1 Cells in Soft
Agar--
Based on the previous results, we became interested in
whether ProT
could induce anchorage-independent growth of Rat-1
cells as determined by growth in soft agar. To this end we used
anchorage-independent growth as another in vitro parameter
to monitor the expression of a fully transformed phenotype (32, 35).
Cells were transfected as described above, with equivalent amounts of
empty pA3M vector, pA3M-ProT
, and pA3M-Ras. 24 h after
transfection, the cells were placed under selection with 800 µg/ml
neomycin in DMEM. After 3 more days of selection, 1.8 × 105 cells were trypsinized, counted and seeded on soft
agar. The cells in soft agar were fed with 1 ml of selection medium
once a week, and plates were observed every 3 days. 22 days after
transfection, colonies were readily apparent in the case of Ras, but
not in control cultures. In the ProT
culture, colonies were apparent but were initially smaller than those observed in the Ras-transfected culture. After 5-6 weeks of selection, ProT
colonies had grown to
the size of the Ras-induced colonies (Fig.
4, panels B and E compared with panels C and
F). Photographs were taken at the end of week 7 for all
plates. Overall, ProT
colonies initially grew slower than
Ras-expressing colonies in soft agar but eventually attained similar
size. Moreover, on average the number of colonies obtained with ProT
was about 50% lower than that seen with Ras but much greater than
vector alone control (Table III).
Additionally, the approximate number of ProT
colonies after 12 weeks
was similar to that of the Ras-transfected cells. Once the ProT
colonies reached a size similar to that of Ras, no other obvious
morphological differences were detected in this assay. Typically, the
vector alone culture showed no sign of colony growth even after
extended incubations in 12 weeks of selection. These results indicate
that ProT
can induce anchorage-independent growth when stably
expressed in Rat-1 cells.
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Fig. 4.
Colony formation in soft agar. Rat-1
cells were transfected with pA3M vector alone, pA3M-ProT , and
pA3M-Ras, followed by selection in 800 µg/ml G418 after 24 h.
48 h later, cells were trypsinized and transferred to soft agar
covered with DMEM. Representative photomicrographs were taken with a
phase contrast microscope (magnification, ×10), after 7 weeks of G418
selection. Photographs of whole plates were taken with a camera
(magnification, ×1.5).
ProT expression in Rat-1 fibroblasts induces colony formation in
soft agar
and Ras were
determined from three different experiments and separate transfections.
Data are presented as mean ± S.D.
from the Transfected
Heterologous Promoter--
To demonstrate that the transformed
phenotype seen in Rat-1 cells transfected with ProT
was due to the
overexpression of ProT
from the heterologous cytomegalovirus
immediate-early promoter, we did Western blots on cells obtained from
individual colonies or foci from the experiments above. Signals of
ProT
were detected using monoclonal antibodies against the Myc tag
fused to ProT
. A specific band was detected in all the
ProT
-transfected cells and was not detected in the cells that were
transfected with vector alone (Fig. 5,
compare lane 1 with lanes
2-5). A smaller band was seen below the specific band in
lanes 2-5, suggesting proteolytic degradation or varying levels of modification of the Myc-tagged ProT
expressed from the heterologous cytomegalovirus immediate-early promoter.
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Fig. 5.
Expression of ProT
in Rat-1 clones. Approximately 1 million clonal Rat-1 cells
transfected with pA3M vector, pA3M-Ras, and pA3M-ProT
(4 different
Rat-1 clones) were lysed in 240 µl of SDS-lysis buffer. Half of the
sample was loaded on to a 15% SDS-polyacrylamide gel, followed by
transfer to 0.22-µm nitrocellulose. Immune detection of
Myc-tagged ProT
from pA3M-ProT
was carried out by incubation with
monoclonal anti-Myc antibody, followed by incubation with anti-mouse
horseradish peroxidase secondary antibody and detection by standard
chemiluminescence. Western blot analysis reveals the presence of a
small (~22 kDa) protein in all four Rat-1 pA3M-ProT
clones, but
not in either pA3M- or pA3M-Ras-transfected Rat-1 cells (compare
lanes 2-5 with lane
1).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
protein remains somewhat obscure. Recent work in our laboratory
has shown that ProT
is involved in transcriptional regulation at the
level of histone acetylation through interaction with p300 and one of
the essential EBV antigen, EBNA3C (36). EBNA3C has been shown to be
required for the in vitro transformation of primary human
lymphocytes by EBV (37) and has been suggested to have oncogenic
potential and deregulatory effects on the cell cycle (38). Moreover,
the in vitro interaction as well as the in vivo
association of ProT
and EBNA3C suggested that ProT
might be a
cellular target of EBNA3C, which results in regulation of cell cycle
events. The additional studies demonstrating that ProT
can interact
with histone H1 in vitro and in cells (30, 31, 36)
strengthened the previous findings that ProT
may have a role in
regulating the assembly of nucleosomes. These studies prompted us to
investigate a possible oncogenic role for ProT
in triggering
cellular growth transformation.
is capable of inducing
not only significant cell proliferation, but also every assayed feature
of transformed cells. Specifically, ProT
expression resulted in the
marked proliferation of transfected Rat-1 cells, elimination of contact
inhibition, and both anchorage- and serum-independent growth of these
cells in vitro. This was shown by the observation and
quantification of multicellular foci, growth in soft agar, and growth
in the presence of low serum. These results were strikingly similar to
those observed upon expression of the known oncoprotein, Ras, whereas
these phenotypes were not observed in Rat-1 clones containing only the
control pA3M vector. Taken together, this indicates that ProT
retains the capacity to function as a transforming oncoprotein.
induces transformation, however, remains
unclear. Although ProT
does not bear sequence homology to any other
known oncogenes, the similarity between the phenotypes induced by
ProT
and Ras is striking. Since ProT
is almost completely localized to the nucleus (39), it is unlikely that it functions by
stimulating a signal transduction cascade from the plasma membrane, as
does the Ras oncoprotein (40, 41). As suggested by recent work in our
laboratory, it is more likely that ProT
functions at the level of
gene transcription by modulating histone acetyltransferase activity in
producing its oncogenic effects (36). Overexpression of ProT
may
thereby result in the transcriptional dysregulation of promoters that
drive expression of cell cycle regulatory proteins.
has structural similarities to other cellular proteins
with acidic domains and includes the high mobility group
proteins, nucleolin, and proliferating cell nuclear antigen (7,
39, 42). The functions of these molecules all relate to the
modification of chromatin structure during activation/repression of
transcription and replication of DNA templates (43). It is possible
that ProT
plays similar roles as an ancillary factor for the basal
transcriptional or replicative machinery in a global sense in lieu of
the fact that ProT
is ubiquitously expressed and up-regulated in
proliferating cells (23, 44, 45). These putative roles correlate with known studies demonstrating that ProT
is required for cell division and is up-regulated in proliferating, transcriptionally active cells
increasing the transition of the cells through G1 (23, 44,
45).
is a transcriptional target of c-Myc
(44). Transcription of the ProT
gene rapidly increases upon Myc
activation probably due to Myc transcriptional activity on an E-box DNA
element located within the first intron of the ProT
gene (25).
c-myc is a well characterized oncogene known to play an
important role in the transformation of primary cells into immortal
cell lines, which can be continually grown in culture (22). The present
work suggests that activation of the ProT
promoter may be
central to the cellular proliferation induced by c-Myc activation.
Although at this time, the exact role of ProT
downstream of c-Myc is
not clear, it is still intriguing to note that ProT
may be a
critical target for c-Myc-mediated induction of cell proliferation and
transformation. ProT
is certainly not the only target for c-Myc
activation, but it is possible that this ubiquitous cellular protein
may be one of the critical targets required to induce the transformed phenotype.
similar to c-Myc. It would therefore
be interesting to note whether or not ProT
could be activated by the
EBV-encoded nuclear proteins required for B cell immortalization. As
stated before, ProT
has been shown to interact with the EBV EBNA3C,
a viral protein required for transformation of primary human B
lymphocytes, and the p300 coactivator/histone acetyltransferase (36).
By interacting with p300, ProT
may regulate transcriptional activity
through modulation of the histone acetylase function of p300. This
would then result in a change in transcriptional activity of promoters
that drive expression of genes regulating proliferation and cell cycle
progression. These studies, which include the identification of the
specific promoters that are affected, are currently under study. It is possible that ProT
may have a role on specific promoters through its
interaction with the basal transcription factors or that it is part of
specific complexes required for activation of specific gene promoters.
The latter is less likely to be true in lieu of the fact that ProT
interacts with coactivators, which include p300 and possibly others and
is generally up-regulated in proliferating cells (36). The fact that
the ProT
/p300 complex is also bound by EBNA3C in EBV-infected cells
does provide some clues. Further studies will also be necessary to
determine what role EBNA3C may play in cell transformation in the
context of ProT
, documented in this study. We are currently
targeting specific cellular pathways utilized by ProT
in an attempt
to determine the mechanism by which ProT
can induce transformation
of mammalian cells in vitro.
expression is induced by
the E2F transcription factor known to regulate cell cycle-related genes
or genes involved in cell proliferation (45). These genes are typically
required for DNA synthesis and cell proliferation (45-47). The
disruption of the E2F·pRb complex due to hyperphosphorylation of the
pRb molecules occurs in late G1 allowing the cells to exit the G1 restriction point (48-51). Moreover, pRb is a known
tumor suppressor or suppressor of cell proliferation and can be
considered to be a regulator of ProT
expression through its
association with E2F, as E2F can activate the ProT
promoter (45).
ProT
expression also correlates with the expression of cyclin B with an increase in S/G2 and reducing as the cell traverses or
enters the G1 phase of a new cycle (45, 52). This
similarity in expression patterns is not fully understood, as studies
are still left to be done that may explain the similarities in terms of
transcription regulation or a functional relationship that is
intimately tied with the cell cycle. Overall, these data indicate that
ProT
is a cell cycle-regulated molecule intricately intertwined in
the processes of DNA synthesis and cell proliferation.
appears to be targeted by at least two
diverse oncogenic stimuli. Specifically, the cellular c-myc oncogene as well as the DNA tumor virus, EBV, both seem to interface directly with ProT
or through its transcriptional regulatory function in the process of cellular transformation (36). The current
study, in which transfected ProT
alone is shown to induce many
features of malignancy in rodent fibro-blasts, serves to corroborate
the importance of this molecule in having a critical role in studies of
basic cancer biology.
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ACKNOWLEDGEMENTS |
---|
We thank George Mosialos for the Rat-1 cell
line and Fernando Dominguez for the ProT construct. We are also
thankful to Bruce Donohoe for assistance with microscopy.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the Leukemia and Lymphoma Society of America and by National Institutes of Health NCI Grant CA072150-01 (to E. S. R.).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.
§ Fellow of the Lady Tata Memorial Trust, London and member of the Medical Scientist Training Program of the University of Michigan Medical School.
A Leukemia and Lymphoma Society of America Scholar. To whom
correspondence should be addressed: Dept. of Microbiology and Immunology and Cellular and Molecular Biology Graduate Program, 1150 W. Medical Center Dr., University of Michigan Medical School, Ann Arbor,
MI 48109-0620. Tel.: 734-647-7296; Fax: 734-764-3562; E-mail:
esrobert@umich.edu.
Published, JBC Papers in Press, October 17, 2000, DOI 10.1074/jbc.M008560200
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ABBREVIATIONS |
---|
The abbreviations used are:
ProT, prothymosin
;
EBV, Epstein-Barr virus;
DMEM, Dulbecco's modified Eagle's
medium.
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