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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Cycle-Dependent PTTG Expression
Human placental JEG-3 cells express PTTG (Fig. 1Go). 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. 1AGo). JEG-3 cells were also synchronized at the G1/S border and at G2/M by treating with aphidicolin and nocodazole, respectively (Fig. 1AGo). PTTG mRNA expression was low at the G1/S border achieved by double thymidine block or by incubation with aphidicolin (Fig. 1BGo). 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. 1CGo). 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.



View larger version (67K):
[in this window]
[in a new window]
 
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.

 
Localization of PTTG Protein in Interphase and Mitosis
Endogenous PTTG protein expression was low in cycling JEG-3 cells (Fig. 1DGo), 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. 2aGo) and the tagged PTTG proteins (Fig. 2Go, 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. 2cGo). 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).



View larger version (81K):
[in this window]
[in a new window]
 
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.

 
Localization of PTTG-EGFP in cycling cells, most of which were in interphase, was confirmed by cell fractionation (Fig. 3Go). 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.



View larger version (33K):
[in this window]
[in a new window]
 
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.

 
In metaphase cells evidenced by aligned chromosomes and characteristic spindles, wild-type PTTG (Fig. 4aGo) and PTTG-FLAG (Fig. 4bGo) 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. 5Go). 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. 5Go, a–c). PTTG-EGFP colocalized with microtubule asters in prophase and prometaphase (Fig. 5Go, a and b). During anaphase, PTTG-EGFP aggregated into distinct granules suggesting a proteasomal degradation process (Fig. 5dGo). No cells in telophase were found to express PTTG-EGFP.



View larger version (21K):
[in this window]
[in a new window]
 
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).

 


View larger version (54K):
[in this window]
[in a new window]
 
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.

 
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 1–3 h before and after release (Figs. 6Go and 7Go). 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. 6Go). The daughter cells were viable because they showed normal morphology hours after mitosis.



View larger version (64K):
[in this window]
[in a new window]
 
Figure 6. Observation of Mitosis of Synchronized, Live Cells

JEG-3 cells were transiently transfected with plasmids encoding EGFP only (panels 1–6), PTTG-EGFP (panels 7–12), or the mutant PTTG-EGFP (panels 13–18). JEG-3 cells were synchronized at the G1/S border by double thymidine block and then released. Live cells were observed every 1–3 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 1–3, 7–9, 13–15) and the corresponding brightfield (panels 4–6, 10–12, 16–18) images were both shown.

 


View larger version (62K):
[in this window]
[in a new window]
 
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 1–3 h for 14 h after release. Shown on the left (panels 1–4) 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 5–8) 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.

 
Surprisingly, most cells (31/46, 67%) expressing wild-type PTTG-EGFP died shortly after cells were released from G1/S block ( Figs. 7–9GoGoGo). TUNEL staining of PTTG-EGFP-expressing dead cells showed that they were apoptotic (Fig. 8Go). In comparison, only 44% (27/61, P < 0.05) of cells expressing EGFP alone died randomly instead of early after release from block (Fig. 9Go). 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. 9Go). Some cells expressing PTTG-EGFP (6/46) or the mutant PTTG-EGFP (2/16) lost EGFP fluorescence while alive during interphase (Fig. 7Go), 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. 9Go), 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. 9Go).



View larger version (46K):
[in this window]
[in a new window]
 
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.

 


View larger version (22K):
[in this window]
[in a new window]
 
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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 (80–100 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 manufacturer’s 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 [{alpha}-32P]dCTP using RadPrime Labeling Kit (Life Technologies, Inc.) according to manufacturer’s 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 {alpha} 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 manufacturer’s 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.


    FOOTNOTES
 
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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Pei L, Melmed S 1997 Isolation and characterization of a pituitary tumor-transforming gene (PTTG). Mol Endocrinol 11:433–441[Abstract/Free Full Text]
  2. Zhang X, Horwitz GA, Prezant TR, Valentini A, Nakashima M, Bronstein MD, Melmed S 1999 Structure, expression, and function of human pituitary tumor-transforming gene (PTTG). Mol Endocrinol 13:156–166[Abstract/Free Full Text]
  3. Dominguez A, Ramos-Morales F, Romero F, Rios RM, Dreyfus F, Tortolero M, Pintor-Toro JA 1998 hpttg, A human homologue of rat pttg, is overexpressed in hematopoietic neoplasms. Evidence for a transcriptional activation function of hPTTG. Oncogene 17:2187–2193[CrossRef][Medline]
  4. Zhang X, Horwitz GA, Heaney AP, Nakashima M, Prezant TR, Bronstein MD, Melmed S 1999 Pituitary tumor transforming gene (PTTG) expression in pituitary adenomas. J Clin Endocrinol Metab 84:761–767[Abstract/Free Full Text]
  5. Heaney AP, Horwitz GA, Wang Z, Singson R, Melmed S 1999 Early involvement of estrogen-induced pituitary tumor transforming gene and fibroblast growth factor expression in prolactinoma pathogenesis. Nat Med 5:1317–1321[CrossRef][Medline]
  6. Heaney AP, Singson R, McCabe CJ, Nelson V, Nakashima M, Melmed S 2000 Pituitary tumor transforming gene in colorectal tumors. Lancet 355:712–715[CrossRef][Medline]
  7. Saez C, Japon MA, Ramos-Morales F, Romero F, Segura DI, Tortolero M, Pintor-Toro JA 1999 hpttg Is overexpressed in pituitary adenomas and other primary epithelial neoplasias. Oncogene 18:5473–5476[CrossRef][Medline]
  8. Wang Z, Melmed S 2000 Pituitary tumor transforming gene (PTTG) transactivating, transforming activity. J Biol Chem 275:7459–7461[Abstract/Free Full Text]
  9. Deleted in proof
  10. Zou H, McGarry TJ, Bernal T, Kirschner MW 1999 Identification of a vertebrate sister-chromatid separation inhibitor involved in transformation and tumorigenesis. Science 285:418–422[Abstract/Free Full Text]
  11. Lieblich JM, Weintraub BD, Krauth GH, Kohler PO, Rabson AS Rosen SW 1976 Ectopic and eutopic secretion of chorionic gonadotropin and its subunits in vitro: comparison of clonal strains from carcinomas of lung and placenta. J Natl Cancer Inst 56:911–917[Medline]
  12. Jameson JL, Jaffe RC, Gleason SL, Habener JF 1986 Transcriptional regulation of chorionic gonadotropin {alpha}- and ß-subunit gene expression by 8-bromo-adenosine 3',5'-monophosphate. Endocrinology 119:2560–2567[Abstract]
  13. Ren SG, Braunstein GD 1991 Insulin stimulates synthesis and release of human chorionic gonadotropin by choriocarcinoma cell lines. Endocrinology 128:1623–1629[Abstract]
  14. Cohen-Fix O, Peters JM, Kirschner MW, Koshland D 1996 Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p. Genes Dev 10:3081–3093[Abstract]
  15. Funabiki H, Yamano H, Kumada K, Nagao K, Hunt T, Yanagida M 1996 Cut2 proteolysis required for sister-chromatid separation in fission yeast. Nature 381:438–441[CrossRef][Medline]
  16. Ramos-Morales F, Dominguez A, Romero F, Luna R, Multon MC, Pintor-Toro JA, Tortolero M 2000 Cell cycle regulated expression, phosphorylation of hpttg proto-oncogene product. Oncogene 19:403–409[CrossRef][Medline]
  17. Kakar SS 1999 Molecular cloning, genomic organization, and identification of the promoter for the human pituitary tumor transforming gene (PTTG). Gene 240:317–324[CrossRef][Medline]
  18. Pei L 1998 Genomic organization and identification of an enhancer element containing binding sites for multiple proteins in rat pituitary tumor-transforming gene. J Biol Chem 273:5219–5225[Abstract/Free Full Text]
  19. Wang Z, Melmed S 2000 Characterization of the murine pituitary tumor transforming gene (PTTG), its promoter. Endocrinology 141:763–771[Abstract/Free Full Text]
  20. Mantovani R 1998 A survey of 178 NF-Y binding CCAAT boxes. Nucleic Acids Res 26:1135–1143[Abstract/Free Full Text]
  21. Kumada K, Nakamura T, Nagao K, Funabiki H, Nakagawa T, Yanagida M 1998 Cut1 is loaded onto the spindle by binding to Cut2 and promotes anaphase spindle movement upon Cut2 proteolysis. Curr Biol 8:633–641[Medline]
  22. Pei L 1999 Pituitary tumor-transforming gene protein associates with ribosomal protein S10 and a novel human homologue of DnaJ in testicular cells. J Biol Chem 274:3151–3158[Abstract/Free Full Text]
  23. King KL, Cidlowski JA 1998 Cell cycle regulation and apoptosis. Annu Rev Physiol 60:601–617[CrossRef][Medline]
  24. Uhlmann F, Lottspeich F, Nasmyth K 1999 Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1. Nature 400:37–42[CrossRef][Medline]