Pituitary Tumor Transforming Gene (PTTG) Regulates Placental JEG-3 Cell Division and Survival: Evidence from Live Cell Imaging
Run Yu,
Song-Guang Ren,
Gregory A. Horwitz,
Zhiyong Wang and
Shlomo Melmed
Division of Endocrinology Cedars-Sinai Research Institute-UCLA
School of Medicine Los Angeles, California 90048
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ABSTRACT
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The pituitary transforming gene, PTTG, is
abundantly expressed in endocrine neoplasms. PTTG has recently been
recognized as a mammalian securin based on its biochemical homology to
Pds1p. PTTG expression and intracellular localization were therefore
studied during the cell cycle in human placental JEG-3 cells. PTTG mRNA
and protein expressions were low at the G1/S
border, gradually increased during S phase, and peaked at
G2/M, but PTTG levels were attenuated as cells
entered G1. In interphase cells, wild-type
PTTG, an epitope-tagged PTTG, and a PTTG-EGFP conjugate all
localized to both the nucleus and cytoplasm, but in mitotic cells, PTTG
was not observed in the chromosome region. PTTG-EGFP colocalized with
mitotic spindles in early mitosis and was degraded in anaphase.
Intracellular fates of PTTG-EGFP and a conjugate of EGFP and a mutant
inactivated PTTG devoid of an SH3-binding domain were observed by
real-time visualization of the EGFP conjugates in live cells. The same
cells were continuously observed as they progressed from
G1/S border to S, G2/M,
and G1. Most cells (67%) expressing PTTG-EGFP
died by apoptosis, and few cells (4%) expressing PTTG-EGFP
divided, whereas those expressing mutant PTTG-EGFP divided.
PTTG-EGFP, as well as the mutant PTTG-EGFP, disappeared after cells
divided. The results show that PTTG expression and localization are
cell cycle-dependent and demonstrate that PTTG regulates endocrine
tumor cell division and survival.
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INTRODUCTION
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Pituitary tumor transforming gene (PTTG) was identified in rat
pituitary tumor cells by differential mRNA display (1). Human PTTG (2, 3) is highly expressed in pituitary tumors (4, 5) and other neoplasms
(3, 6, 7). Levels of PTTG expression positively correlate with
pituitary tumor invasiveness (4) and are induced by estrogen (5). The
mechanism of PTTG action is not clear. PTTG up-regulates basic
fibroblast growth factor secretion (2) and transactivates DNA
transcription (Refs. 3, 8 and Horwitz, G. A., Z. Wong, X. Zhang, and
S. Melmed, unpublished). Recently, PTTG protein has been recognized as
a mammalian securin protein that maintains binding of sister chromatids
during mitosis (10). At the end of metaphase, securin is degraded by an
anaphasepromoting complex, releasing tonic inhibition of separin, which
in turn mediates degradation of cohesins, the proteins that hold sister
chromatids together. Overexpression of a nondegradable PTTG disrupts
sister chromatid separation (10).
Since PTTG was identified, its cellular characteristics have
not been studied. To address the mechanism of PTTG action, we studied
PTTG expression and intracellular localization during the cell cycle in
human placental JEG-3 cells, which are among the few available human
endocrine cells in culture. The effects of PTTG expression on the cell
cycle were also addressed. JEG-3 cells secrete hCG both basally and in
response to a variety of stimulants (11, 12, 13). Our results show that
PTTG expression and localization are cell cycle dependent and
demonstrate that PTTG regulates placental tumor cell division and
survival.
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RESULTS
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Cell Cycle-Dependent PTTG Expression
Human placental JEG-3 cells express PTTG (Fig. 1
). After JEG-3 cells were synchronized
at the G1/S border with double thymidine block
and then released, they gradually entered S,
G2/M, and G1 over 15 h
(Fig. 1A
). JEG-3 cells were also synchronized at the
G1/S border and at G2/M by
treating with aphidicolin and nocodazole, respectively (Fig. 1A
). PTTG
mRNA expression was low at the G1/S border
achieved by double thymidine block or by incubation with aphidicolin
(Fig. 1B
). When cells were released, PTTG mRNA increased through the S
phase and plateaued at G2/M (2.0 ± 0.3-fold
over G1/S). Cells synchronized by nocodazole also
expressed higher mRNA levels than did cycling cells. PTTG protein
expression followed a similar cycle-dependent course (Fig. 1C
). PTTG
protein levels assessed by Western blotting were very low in cells at
the G1/S border, increased through S phase, and
peaked at G2/M (2.4 ± 0.6-fold over
G1/S). When cells divided, PTTG protein levels
again decreased.

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Figure 1. Cell-Cycle Dependent Expression of PTTG
JEG-3 cells were synchronized at G1/S border by double
thymidine block and then released. JEG-3 cells were also blocked at the
G1/S border by aphidicolin, or at G2/M by
nocodazole. A, Phase components measured by flow cytometry after
release from double thymidine block, or after treatments with
aphidicolin or nocodazole. B, Corresponding PTTG mRNA measured by
Northern blotting. Equal amounts of total RNA (10 µg) were loaded in
each lane. C, PTTG protein after release from double thymidine block
measured by Western blotting using a rabbit antiserum against PTTG.
Equal amounts of protein (150 µg) were loaded in each lane. D, JEG-3
cells were transiently transfected with a pCIneo plasmid encoding PTTG
and endogenous and overexpressed PTTG in cycling JEG-3 cells were shown
in Western blot stained with rabbit anti-PTTG. UT, Untransfected, and
T, transfected with PTTG. Experiments were repeated two to three times
with similar results.
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Localization of PTTG Protein in Interphase and Mitosis
Endogenous PTTG protein expression was low in cycling JEG-3 cells
(Fig. 1D
), and no significant results were obtained after
immunofluorescent staining of those cells with rabbit anti-PTTG serum.
To study PTTG localization, JEG-3 cells were transfected with plasmids
encoding wild-type PTTG, a FLAG epitope-tagged PTTG (PTTG-FLAG), or a
conjugated PTTG and EGFP protein (PTTG-EGFP). Intracellular
localization of wild-type PTTG was followed by immunofluorescent
staining using a rabbit anti-PTTG serum. No significant staining was
found when transfected cells were stained with preimmune serum or when
untransfected cells were stained with antiserum. Localization of
FLAG-tagged PTTG was revealed with immunofluorescent staining using the
M2 monoclonal antibody. Again, no significant immunoreactivity was
observed when transfected cells were stained with an irrelevant
monoclonal antibody or when untransfected cells were stained with the
M2 antibody. The PTTG-EGFP conjugate was directly visualized after
fixing. JEG-3 cells were also stained with Hoechst 33258 to delineate
nuclear structures. In cycling JEG-3 cells, localizations of the three
PTTG protein constructs were similar. During interphase, wild-type PTTG
protein (Fig. 2a
) and the tagged PTTG
proteins (Fig. 2
, b and c) were evident throughout the cell, but PTTG
was more concentrated in the nucleus than in the cytoplasm. Both the
PTTG signals as well as DNA appeared distinct from the nucleoli. In
some cells, PTTG protein also localized on the plasma membrane (found
in <5% of transfected cells) (Fig. 2c
). Localization of PTTG protein
constructs was similar regardless of their respective expression level.
Similar results were obtained when PTTG-EGFP was expressed in a variety
of other cell lines including 3T3 murine fibroblast, GH3 and AtT20 rat
pituitary tumor, SKOV-3 human ovarian cancer, MCF-7 human breast
cancer, and COS-7 monkey kidney cells (data not shown).

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Figure 2. Localization of PTTG in Interphase
JEG-3 cells were transfected with plasmids encoding PTTG (a), PTTG-FLAG
(b), or PTTG-EGFP (c). PTTG or PTTG-FLAG was visualized by
immunofluorescent staining, and PTTG-EGFP was visualized directly
(left). Cells were also stained with Hoechst 33258
(middle) to highlight the nuclei and chromosomes and to
determine the cell cycle. The images of cells expressing PTTG,
PTTG-FLAG, or PTTG-EGFP were overlaid with images of cells stained with
Hoechst (right). Green, PTTG or
PTTG-EGFP; red: PTTG-FLAG; and blue, DNA.
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Localization of PTTG-EGFP in cycling cells, most of which were in
interphase, was confirmed by cell fractionation (Fig. 3
). JEG-3 cells were transfected with
plasmids encoding EGFP alone or PTTG-EGFP. EGFP and PTTG-EGFP were
determined in cytosolic and nuclear fractions by Western blotting. EGFP
was detected predominantly in the cytosol while PTTG-EGFP localized
mostly in cell nuclei. The cytosolic fraction of cells expressing
PTTG-EGFP contained two discrete EGFP antibody-reactive proteins in
addition to EGFP, probably due to proteolysis of PTTG-EGFP. The purity
of the cytosolic and nuclear fractions was confirmed by Western blot
using cytosolic marker protein tubulin and nuclear marker protein
lamin. No similar visualizing attempts were made on mitotic cells
because they do not exhibit defined nuclei.

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Figure 3. Localization of PTTG-EGFP Determined by Cell
Fractionation
JEG-3 cells were transfected with plasmids encoding EGFP only or
PTTG-EGFP. A, Cytosolic (C) and nuclear (N) fractions were collected
and EGFP or PTTG-EGFP in each fraction was examined by Western blotting
using antibody against EGFP. B, The purity of the cytosolic and nuclear
fractions was verified with antibodies to tubulin and nuclear lamin.
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In metaphase cells evidenced by aligned chromosomes and characteristic
spindles, wild-type PTTG (Fig. 4a
) and
PTTG-FLAG (Fig. 4b
) were not visible in the chromosome region but
remained elsewhere. Costaining of PTTG-FLAG and spindles was not
attempted because antibodies to both of them were of mouse origin. In
these experiments, PTTG-EGFP transfection efficiency was highest and
facilitated sufficient numbers of mitotic cells expressing PTTG-EGFP
available for subsequent following (Fig. 5
). Mitotic spindles were visualized by
utilizing antibodies to tubulin. Although PTTG-EGFP was well visualized
from prophase to metaphase, it was distinct from chromosomes (Fig. 5
, ac). PTTG-EGFP colocalized with microtubule asters in prophase and
prometaphase (Fig. 5
, a and b). During anaphase, PTTG-EGFP aggregated
into distinct granules suggesting a proteasomal degradation process
(Fig. 5d
). No cells in telophase were found to express PTTG-EGFP.

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Figure 4. Localization of PTTG in Mitosis
JEG-3 cells were transfected with plasmids encoding PTTG (a), or
PTTG-FLAG (b). PTTG or PTTG-FLAG was visualized by immunofluorescent
staining (PTTG). Cells were stained with Hoechst 33258 to highlight the
mitotic nuclei (DNA). Wild-type PTTG-transfected cells were also
stained with antibodies to tubulin (spindle). a, Images of PTTG, DNA,
and mitotic spindle were overlaid. Green, PTTG;
red, mitotic spindles; and blue, DNA. b,
Images of PTTG-FLAG and DNA were overlaid. Red,
PTTG-FLAG; and blue, DNA (looks purple
because of some mixing with red).
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Figure 5. Localization of PTTG-EGFP in Mitosis
JEG-3 cells were transfected with plasmids encoding PTTG-EGFP.
PTTG-EGFP was visualized directly (left). Mitotic
spindles were visualized by immunofluorescent staining of tubulin
(middle left). Cells were stained with Hoechst 33258 to
highlight the stage in mitosis (middle right). Images of
PTTG-EGFP, mitotic spindle, and chromosome were overlaid
(right). Green, PTTG-EGFP;
red, mitotic spindles; and blue, DNA.
Orange or yellow indicates where
green and red colocalized. a, Prophase;
b, prometaphase; c, metaphase; and d, anaphase.
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Observation of PTTG-EGFP in Live, Synchronized Cells
To follow the fate of PTTG more closely and to study cellular
effects of overexpressing PTTG, JEG-3 cells were transfected with
plasmids encoding EGFP alone, PTTG-EGFP, and a mutant PTTG-EGFP. This
mutant PTTG is mutated in the SH3-binding region (P163A, P170L, P172A,
and P173L) and is devoid of transforming activity (2). Transfected
cells were synchronized at the G1/S border and
released into normal cell cycle, and the same group of live cells were
observed every 13 h before and after release (Figs. 6
and 7
).
Thus, EGFP, PTTG-EGFP, and mutant PTTG-EGFP were visualized in a
real-time manner in live single cells for up to 14 h. EGFP alone
was evenly distributed throughout the cells. PTTG-EGFP and mutant
PTTG-EGFP were both evident largely in the nuclei, suggesting that the
SH3-binding domain was not responsible for the nuclear localization. In
cells expressing EGFP alone, EGFP remained intact during and after
mitosis; however, in cells expressing wild-type or the mutant
PTTG-EGFP, EGFP fluorescence was dissipated immediately after mitosis
in daughter cells (Fig. 6
). The daughter cells were viable because they
showed normal morphology hours after mitosis.

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Figure 6. Observation of Mitosis of Synchronized, Live Cells
JEG-3 cells were transiently transfected with plasmids encoding EGFP
only (panels 16), PTTG-EGFP (panels 712), or the mutant PTTG-EGFP
(panels 1318). JEG-3 cells were synchronized at the G1/S
border by double thymidine block and then released. Live cells were
observed every 13 h for 14 h after release. Cells that divided
were shown before (panels 1 and 4; 7 and 10; 13 and 16), during (panels
2 and 5; 8 and 11; 14 and 17), and after mitosis (panels 3 and 6; 9 and
12; 15 and 18). Fluorescent (panels 13, 79, 1315) and the
corresponding brightfield (panels 46, 1012, 1618) images were
both shown.
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Figure 7. Cell Death and Degradation of PTTG-EGFP
JEG-3 cells were transiently transfected with plasmids encoding
PTTG-EGFP. JEG-3 cells were synchronized at the G1/S border
by double thymidine block and then released. Live cells were observed
every 13 h for 14 h after release. Shown on the
left (panels 14) are the same cells before (panels 1
and 3) and 2 h after (panels 2 and 4) release from block. One of
the two cells expressing PTTG-EGFP died after release. Shown on the
right (panels 58) are the same cells 4 h (5 7 ) and 7 h (6 8 ) after release. Two of the three cells
expressing PTTG-EGFP degraded PTTG-EGFP. Fluorescent (panels 1, 2, 5,
and 6) and the corresponding brightfield (panels 3, 4, 7, and 8) images
were both shown.
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Surprisingly, most cells (31/46, 67%) expressing wild-type PTTG-EGFP
died shortly after cells were released from G1/S
block (
Figs. 79

). TUNEL staining of PTTG-EGFP-expressing dead cells
showed that they were apoptotic (Fig. 8
).
In comparison, only 44% (27/61, P < 0.05) of cells
expressing EGFP alone died randomly instead of early after release from
block (Fig. 9
). This background cell
death may be the result of double thymidine block. About 30% (5/16,
fewer cells were observed due to low transfection efficiency) of cells
expressing mutant PTTG-EGFP died early after cells entered S phase. No
cells expressing EGFP alone demonstrated EGFP degradation, indicating
that EGFP itself is stable (Fig. 9
). Some cells expressing PTTG-EGFP
(6/46) or the mutant PTTG-EGFP (2/16) lost EGFP fluorescence while
alive during interphase (Fig. 7
), suggesting that both the wild-type
and mutant PTTGs are also degraded in interphase. Only 4% of cells
(2/46) expressing wild-type PTTG-EGFP eventually divided (Fig. 9
), and
mitosis was delayed when compared with cells expressing EGFP only, in
which 8 of 61 (13%) divided. On the contrary, more cells (7/16, 44%)
expressing mutant PTTG-EGFP divided and mitosis appeared to occur
slightly earlier than in cells expressing EGFP (Fig. 9
).

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Figure 8. Apoptosis of PTTG-EGFP-Expressing Cells
JEG-3 cells transiently expressing PTTG-EGFP were synchronized at the
G1/S border by double thymidine block then released. Cells
were fixed 3.5 h after release and apoptotic cells were identified
with a TUNEL staining kit using rhodamine-labeled UTP as substrate.
Shown is one PTTG-EGFP expressing cell that is also positive for TUNEL
staining.
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Figure 9. Effects of PTTG on Cell Survival and Division
JEG-3 cells were transiently transfected with plasmids encoding EGFP,
PTTG-EGFP, or the mutant PTTG-EGFP. JEG-3 cells were synchronized at
G1/S border by double thymidine block then released. Live
cells were observed every 1 to 3 h for 14 h after release. A,
Total number of cells that died, remained unchanged, degraded EGFP or
EGFP conjugates, or divided during this 14 h. B, Number of cells
that died over each observation. C, Number of cells that divided over
each observation. Number of cells observed: 61 for EGFP, 46 for
PTTG-EGFP, and 16 for the mutant PTTG-EGFP.
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DISCUSSION
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In this report, we characterize the cellular properties of PTTG in
a human placental JEG-3 tumor cell line. JEG-3 cells express endogenous
PTTG, making them a suitable human endocrine cell model for PTTG study.
We first demonstrated that, in cell populations, expressions of PTTG
mRNA and protein were both highly dependent on the cell cycle, highest
at the G2/M but decreased in the subsequent
G1 phase. At the single-cell level, intracellular
PTTG imaging showed that PTTG was degraded in anaphase, and daughter
cells express little PTTG. Degradation of PTTG protein upon cell
division probably occurs by a proteosomal mechanism as suggested by the
PTTG morphology during anaphase. PTTG contains a D-box, which is
required for ubiquitin-mediated PTTG proteolysis (10). This cell
cycle-dependent PTTG expression is consistent with that of a securin
(10, 14, 15, 16). The human (17), rat (18), and mouse (19) PTTG promoters
contain a conserved region that harbors several motifs such as CCAAT,
which confer cell cycle-dependent expression (20). Thus, the mechanism
of cell cycle-dependent PTTG mRNA expression probably lies at the
transcriptional level. Interestingly, PTTG has also been shown to be
serine phosphorylated during mitosis (16).
We have also detailed the subcellular localization of PTTG during
interphase and in different phases of mitosis. The subcellular
localization of PTTG is similar to that of budding yeast securin Pds1
(14) and fission yeast securin Cut2 (15). Intracellular PTTG
localization was predominantly nuclear in interphase with significant
expression in the cytoplasm, but localized to mitotic spindles during
early mitosis. Binding of securin to mitotic spindles is important for
normal anaphase (21). We recognize that our imaging results depend on
overexpressing PTTG proteins since endogenous PTTG protein level is low
in JEG-3 cells. It is unlikely, however, that the observed localization
of PTTG is due to overexpression, because erroneous localization as a
result of overexpression usually occurs in the endoplasmic reticulum
and the Golgi apparatus, organelles in which PTTG was not seen.
Furthermore, similar results were derived from imaging of three
different PTTG protein constructs as well as from cell fractionation
experiments. The dual intracellular localization of PTTG is consistent
with PTTG functions. As a securin (10), PTTG is required in the nucleus
to regulate sister chromatid binding, and PTTG binds to a ribosomal
protein DnaJ, which presumably is situated in the cytoplasm (22). Based
on these lines of evidence, it appears that the results shown here
faithfully describe the physiological subcellular localization of PTTG
protein. It is not clear how PTTG is targeted to the nucleus. Although
PTTG does not have an obvious nuclear localization signal, its small
size (apparent mobility
28 kDa) may permit it to enter the nucleus.
Apparently, PTTG is not excluded from the nucleus because PTTG-EGFP is
also predominantly nuclear. Our results are in contrast to previous
reports suggesting that immunoreactive PTTG is localized predominantly
in the cytoplasm (3, 4, 7). Although this difference may be due to
different cell and tissue preparations used in these studies, we have
now shown similar subcellular localization of PTTG-EGFP in eight
different cell lines.
Functionally, PTTG overexpression inhibited mitosis as few cells
overexpressing PTTG divided and cell division appeared delayed. The
inhibitory effect of PTTG on mitosis is expected of a securin because
the cell requires more time to degrade overexpressed PTTG before
anaphase. In contrast to yeast cells that survive without sister
chromatid separation (15), most human placental cells overexpressing
PTTG died by apoptosis. Overexpressing PTTG may cause general cell
cycle disruption or may induce transcription of apoptosis-promoting
genes. It is well documented that cell cycle disruption may cause
apoptosis (23). Our results are consistent with those of Uhlmann
et al. (24) who showed that cells expressing an uncleavable
form of a cohesin subunit die. Apoptosis induced by PTTG and the fewer
and delayed mitoses provide an explanation for the finding that
overexpressing PTTG inhibits cell proliferation (1).
This study again highlights the functional importance of the
SH3-binding domain in the C terminus of PTTG protein. Mutant PTTG
lacking an SH3-binding domain is devoid of transforming activity (2,
and Horwitz, G. A., Z. Wang, X. Zhang, and S. Melmed, unpublished),
suggesting that critical interactions between PTTG and proteins
containing SH3 domains are responsible for PTTG function. Our results
suggest that the SH3-binding domain is not important for nuclear or
cytosolic localization because the localization of this mutant PTTG was
similar to that of wild-type PTTG. Since mutant PTTG facilitated
mitosis as evidenced by more frequent and earlier mitosis, probably due
to easier separation of sister chromatids, the SH3-binding domain may
also be important for the securin function of PTTG.
Several lines of evidence from our study strongly indicate that PTTG is
a securin in JEG-3 cells. Based on the securin function of PTTG, we
propose that chromosomal instability and induction of aneuploidy may be
a mechanism of PTTG action. By keeping sister chromatids held
abnormally tight, overexpression of PTTG may enable daughter cells to
either gain or lose one or more chromosomes. In both cases, the
daughter cell may become tumorous.
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MATERIALS AND METHODS
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Cell Synchronization
Human placental choriocarcinoma JEG-3 cells were grown in DMEM
plus 10% FBS. JEG-3 cells were grown to halfconfluency, treated
with 2 mM thymidine overnight, and were grown in normal
medium for 10 h. When required, cells were transfected using
FuGene (Roche Molecular Biochemicals, Indianapolis,
IN) during this growth, and again treated with 2 mM
thymidine overnight. At this stage, cells were blocked at the
G1/S border. Cells were released from the block
by growing in normal medium supplemented with 10 µM
deoxycytidine, and cell cycle progression was confirmed by
fluorescence-activated cell sorting. Cells were also
synchronized at the G1/S border by incubating
with aphidicolin (Calbiochem, La Jolla, CA, 2 µg/ml) for
12 h, and at G2/M by incubating with
nocodazole (Calbiochem, 150 ng/ml) for 16 h.
Plasmids and Transfection
Wild-type human PTTG and an SH3-binding domain-mutated PTTG (2)
from a pCIneo vector were subcloned into an
EcoRI/BamHI site in a pEGFPN3 vector
(CLONTECH Laboratories, Inc. Palo Alto, CA). Wild-type
PTTG was also subcloned into an EcoI/XhoI site in a pCMVtag4
vector (Stratagene, La Jolla, CA). In both cases, EGFP and
the FLAG epitope were fused in frame to the PTTG C terminus and the
fusion proteins were termed PTTG-EGFP and PTTG-FLAG, respectively.
Cells were transfected with FuGene (Roche Molecular Biochemicals), and plasmid-FuGene complex was directly
added to cells grown in normal medium.
Preparation of Rabbit Antibody to PTTG
Polypeptide, PLKOKOPSFSAKKM-TEKTVKA (80100 in human PTTG
protein), was used to generate rabbit antiserum against human PTTG
protein. This antiserum recognized purified PTTG protein. The antiserum
and an anti-EGFP antibody detected the same PTTG-EGFP band (
55 kDa)
by Western blot. The antiserum recognized a predominant 28-kDa protein
in lysates of cells overexpressing PTTG.
Northern Blot Analysis
Total RNA from treated JEG-3 cells was extracted using Trizol
Reagent (Life Technologies, Inc., Gaithersburg, MD)
according to manufacturers protocol. Isolated RNA was precipitated,
washed, and denatured as described previously (2). Separated RNA was
transferred to Hybond-N nylon membrane (Amersham International, Buckinghamshire, UK). The membrane was
cross-linked and prehybridized at 68 C, and hybridized with
approximately 107 cpm
32P-labeled human PTTG cDNA in the presence of
100 µg/ml salmon sperm DNA (Stratagene). The entire
human PTTG cDNA coding region was labeled with
[
-32P]dCTP using RadPrime Labeling Kit
(Life Technologies, Inc.) according to manufacturers
protocol. Blots were then exposed to x-ray film (Eastman Kodak Co., Rochester, NY). A predominant 0.9-kb band was recognized by
this probe as was previously shown (2).
Cell Fractionation
JEG-3 cells were trypsinized and incubated in TM buffer (10
mM Tris, 2 mM MgCl2, pH
7.4) containing 500 nM phenylmethylsulfonyl fluoride for 10
min on ice. Triton x 100 (0.5% final concentration) was added
and cells were incubated for a further 5 min on ice. Cell suspension
was passed through a 22-gauge needle eight times and centrifuged at
800 x g at 4 C for 10 min. The pellet nuclear fraction was
examined with microscopy for purity. Antibodies to
tubulin and
nuclear lamin B (Calbiochem) were used in Western blot to
verify the purity of cytosolic and nuclear fractions.
Western Blotting
Cells were lysed in SDS-PAGE sample buffer (100 µl per 35-mm
dish). Cell lysate (2 to 20 µl) was run on 10% SDS PAGE, and
proteins were transferred to polyvinylidene fluoride membrane
and incubated with rabbit antiserum for PTTG (1:1,000) or monoclonal
EGFP antibody (CLONTECH Laboratories, Inc. 1:1,000) at 4 C
overnight followed by incubation with peroxidase-linked secondary
antibody (Amersham Pharmacia Biotech, Arlington Heights,
IL; 1:5,000). Blots were visualized with ECL (enhanced
chemiluminescence) reagents (Amersham Pharmacia Biotech).
Immunofluorescent Staining
JEG-3 cells were rinsed with PBS, fixed with 4%
paraformaldehyde in PBS, and permeabilized with blocking buffer (PBS
with 5% FBS and 0.6% Tween 20). Primary antibody, rabbit polyclonal
anti-PTTG, or mouse M2 monoclonal anti-FLAG (Stratagene),
was added at 1:1,000 in the blocking buffer and incubated for 2 h.
Cells were washed and incubated with fluorescein
isothiocyanate-labeled swine anti-rabbit IgG (DAKO Corp. A/S, Glostrup, Denmark) or TRITC-labeled goat
antimouse IgG (Molecular Probes, Inc., Eugene, OR) (1:500)
for 30 min. Cells were finally stained with Hoechst 33258
(Molecular Probes, Inc., 1:10,000) for 5 min. Mitotic
spindles were stained with a monoclonal tubulin antibody
(Calbiochem). Samples were washed and kept in mowiol-based
mounting medium. Cells expressing EGFP conjugate were fixed and stained
with Hoechst 33258 and directly visualized. Apoptotic cells were
identified with a TUNEL staining kit using rhodamine-labeled UTP as
substrate (Roche Molecular Biochemicals).
Staining was carried out according to the manufacturers recommended
protocol. Apoptotic cells were visualized with rhodamine filters.
Fluorescence Microscopy
Live or immunochemically stained cells were visualized on a
TE200 inverted epifluorescence microscope (Nikon,
Melville, NY) equipped with relevant fluorescence filters. Digital
images were acquired by a SPOT CCD camera (Diagnostic Instruments,
Sterling Heights, MI) and analyzed by ImagePro software (Media
Cybernetics, Bothell, WA). Serial observation of live cells was
achieved by growing cells in a gridded flask and directly visualized
with a 10x lens and an extra long working distance 40x lens. Several
hundred cells were observed in each of the two to seven staining
experiments and representative cells were depicted in the figures.
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FOOTNOTES
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Address requests for reprints to: Shlomo Melmed, Academic Affairs, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, California 90048. E-mail: melmed{at}csmc.edu
Supported by NIH Grant CA-75979 and the Doris Factor Molecular
Endocrinology Laboratory.
Received for publication March 15, 2000.
Revision received April 20, 2000.
Accepted for publication April 26, 2000.
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REFERENCES
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