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[Cancer Research 62, 3861-3867, July 1, 2002]
© 2002 American Association for Cancer Research

Tumor Biology

SYT-SSX Is Critical for Cyclin D1 Expression in Synovial Sarcoma Cells

A Gain of Function of the t(X;18)(p11.2;q11.2) Translocation1

Yuntao Xie, Björn Skytting, Gunnar Nilsson, Alessandra Gasbarri, Karl Haslam, Armando Bartolazzi, Bertha Brodin, Nils Mandahl and Olle Larsson2

Departments of Oncology and Pathology [Y. X., K. H., A. B., B. B., O. L.] and Orthopedics [G. N.], Karolinska Hospital, SE-171 76 Stockholm, Sweden; Department of Orthopedics, Stockholm Soder Hospital, SE-118 83 Stockholm, Sweden [B. S.]; Department of Clinical Genetics, University Hospital, SE-221 85 Lund, Sweden [N. M.]; Department of Immunology, Regina Elena Cancer Institute, 00158 Rome, Italy [A. G.]; Department of Oncology, Zhongnan Hospital, Wuhan University, 430071 Wuhan, People’s Republic of China [Y. X.]; and Department of Biological Science, Dublin Institute of Technology, Dublin 2, Ireland [K. H.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The SYT-SSX fusion gene has been implicated in the malignant tumor cell growth of synovial sarcoma, but the underlying molecular mechanisms are still poorly understood. Here we demonstrate that SYT-SSX is critical for the protein level of cyclin D1 in synovial sarcoma cells. Antisense oligonucleotides to SYT-SSX mRNA rapidly and drastically decreased cyclin D1 and subsequently inhibited cell growth. This effect is specific for SYT-SSX, without involving the wild-type genes SYT or SSX. The decrease in cyclin D1 expression, which occurred shortly after inhibition of SYT-SSX expression, was found to be primarily dependent on an increased degradation of the cyclin D1 protein, as assessed by pulse-chase experiments using [35S]methionine. Furthermore, transfection of mouse fibroblasts with SYT-SSX cDNA increased the stability of cyclin D1. Because treatment with a proteasome inhibitor restored cyclin D1 expression, it seems like SYT-SSX interferes with ubiquitin-dependent degradation of cyclin D1. However, SYT-SSX-regulated cyclin D1 expression was proven to be independent of the glycogen synthetase kinase-3ß pathway. Taken together, our study provides evidence that SYT-SSX stabilizes cyclin D1 and is critical for cyclin D1 expression in synovial sarcoma cells. SYT-SSX-dependent expression of cyclin D1 may be an important mechanism in the development and progression of synovial sarcoma and also raises the possibility for targeted therapy.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Synovial sarcoma, which is a highly malignant soft tissue tumor occurring mainly in young and middle-aged adults (1) , is characterized by the translocation t(X;18)(p11.2;q11.2) (2) . This translocation results in a fusion between the SYT gene on chromosome 18 and SSX1, SSX2, or SSX4 on the X chromosome, forming the new chimeric genes SYT-SSX1, SYT-SSX2, or SYT-SSX4 (3, 4, 5, 6) .

The normal SYT gene is expressed in a wide variety of cell types and normal tissues. The gene product is localized in the cell nucleus and contains transcription-activating regions, but no DNA-binding domains (7 , 8) . Recently, it was reported that SYT interacts with p300 in the regulation of cell adhesion (9) . The SSX genes have a very restricted expression in normal tissues but are widely expressed in certain malignant cells (10) . The COOH-terminus contains a repression domain (SSXRD). Thus, the SYT-SSX fusion protein contains both potential transcriptional and repressor domains. However, the function of SYT-SSX is still unclear. A recent study by Nagai et al. (11) provided evidence that SYT-SSX has a malignant transforming ability and that it is functionally linked to the transcriptional regulatory machinery involving hBRM/hSNF2a. Other results have shown a link between SYT-SSX and tumor cell proliferation in synovial sarcoma (12 , 13) , indicating that the fusion gene may lead to uncontrolled cell growth. Because uncontrolled cell growth is a crucial event in tumor transformation and progression (14) , a functional interaction between SYT-SSX and the cell cycle machinery would constitute an important mechanism in development of synovial sarcoma.

AS3 ODNs have emerged as an important tool in gene function and target validation both in vitro and vivo (15) . AS ODNs bind to complementary sequences of their targeted mRNA and catalyze the degradation of the mRNA molecule by the nuclease RNase H (16) . In this study, we used the AS strategy to investigate whether SYT-SSX has a regulatory influence on various cell cycle proteins. Our results demonstrate that SYT-SSX is critical for the expression of cyclin D1 in synovial sarcoma cells, seemingly by protecting it from ubiquitin-dependent degradation. Because overexpressed cyclin D1 has been implicated in the transformation and development of many types of cancer (17, 18, 19) , our present findings may be valuable for understanding the pathogenic role of the SYT-SSX fusion gene in synovial sarcoma. Furthermore, it opens the possibility of using SYT-SSX as a therapeutic target.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Proteasome inhibitor ALLN, PI3K inhibitor LY294002, and lithium chloride (LiCl) were purchased from Sigma (St. Louis, MO).

Cell Lines.
The synovial sarcoma cell line 1273/99, expressing the SYT-SSX2 transcript, was established as described previously (20 , 21) . This cell line was cultured in Ham’s F-12 medium supplemented with 20% FBS. The synovial sarcoma cell line CME-1, expressing SYT-SSX2, was a kind gift from Dr. M. Pierotti (Instituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy). SYT-SSX and mock-transfected 3Y1 rat fibroblasts were kind gifts from Dr. K. Nagashima (Hokkaido University School of Medicine, Sapporo, Japan). The NIH3T3 cell line, the Ewing’s sarcoma cell line RD-ES, and the melanoma cell line MEL-28 were obtained from the American Type Culture Collection (Manassas, VA). The melanoma cell line DFW was kindly supplied by Dr. Rolf Kiessling (Karolinska Hospital, Stockholm, Sweden). All these cell lines were cultured in RPMI 1640 supplemented with 10% FBS, with the exception of the NIH3T3 and 3Y1 cell lines, which were cultured in Ivory MEM and DMEM supplemented with 10% FBS, respectively. All cells were maintained at 37°C in a humidified 5% CO2 atmosphere.

Semiquantitative RT-PCR.
Total RNA was isolated using the Qiagen RNeasy kit (Qiagen, Hilden, Germany). The RT-PCR method used for semiquantitative analysis has been described elsewhere (22) . In brief, 500 ng of total RNA were reversed to cDNA using random primers. The same amount of cDNA was loaded to perform PCR for analysis of SYT-SSX, SYT, SSX, and cyclin D1 transcripts. Actin was used as an internal control. The primers used for this study were as follows: (a) SYT-SSX, 5'-AGACCAACACGCCTGGACCA-3' and 5'-CTCGTCATCTTCCTCAGGGTC-3'; (b) wt SYT, 5'-TACTCAGGCCAGGAAGACTA-3' and 5'-CTGTCCAATGTTGCCATCTA-3'; (c) wt SSX, 5'-AGGTTGAACATCCTCAGATG-3' and 5'-CTCGTCATCTTCCTCAGGGTC-3'; (d) cyclin D1, 5'-TCTAAGATGAAGGAGACCATC-3' and 5'-GCGGTAGTAGGACAGGAAGTTGTT-3'; and (e) actin, 5'-CATGCCATCCTGCGTCTGGAC-3' and 5'-CACGGAGTACTTGCGCTCAGGAGG-3'. Amplification was performed at 94°C for 30 s, at 60–66°C for 30 s, and at 72°C for 30 s for 26 or 28 cycles, and a final elongation was performed for 10 min. The PCR products were detected by ethidium bromide staining on a 2% agarose gel.

AS Assays.
Four AS ODNs, corresponding sense ODNs, and two mismatch AS ODNs were designed and purchased from Life Technologies, Inc.: (a) AS1 (5'-CCACAGACATGTTGCCGCCCATCCA-3') covers the start ATG codon of the SYT part of the SYT-SSX fusion transcript; (b) mismatch-AS1 (5'-CAGCCAACATGCCGTTGCCCACCTA); (c) AS2 covers the breakpoint region of SYT-SSX fusion genes (5'-TCTTGGGCATGATCTGGTCATATCC-3'); (d) mismatch-AS2 (5'-TCGGGTTCATGTGCATGTCACATTC); (e) AS3 is specific for wt SYT (5'-TCACTGCTGGTAATTTCCATACTGT-3'), starting from position 1155 of SYT mRNA, which is not involved in the SYT-SSX fusion gene; and (f) AS4 is specific for the SSX part of the SYT-SSX fusion gene (5'-TTCTCTCACGCAGTCTGTGGGTCCA-3'), starting at position 581 of SSX mRNA. All ODNs used were phosphorothiolated. The AS experiments were optimized and carried out carefully following the manufacturer’s instructions. Before starting the experiments, the optimal concentrations of Lipofectin (Life Technologies, Inc.) and ODNs were determined for each cell line. Cells were seeded in 60-mm culture dishes containing 3 ml of medium. When cells reached 40–60% confluence, the AS experiments were performed. The 1273/99, RD-ES, MEL-28, DFW, and NIH3T3/SYT-SSX cells were treated with 0.5 µM AS ODN in 10 µg/ml Lipofectin and medium supplemented with 10% FBS (1273/99) and 5% FBS (other cells). Whereas 1.0 µM AS ODN in 10 µg/ml Lipofectin and medium with 5% FBS was optimal for the CME-1 cell line. For each AS ODN experiment, there was an untreated control (C), a Lipofectin control (L), Lipofectin with AS ODN (AS), and Lipofectin with sense ODN (S) or mismatch AS ODN. The cells were harvested after the indicated times.

Western Blotting.
Cells were lysed in lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.5% Triton-X-100 supplemented with protease inhibitor tablet] and prepared for gel electrophoresis and Western blotting as described previously (22) . The immunoblots were probed with the following primary antibodies: anti-actin (Sigma); anti-GSK-3ß (BD Transduction Laboratories, Franklin Lakes, NJ); anti-Akt, anti-pAkt, and anti-pGSK-3ß (New England Biolabs, Beverly, MA); and anti-cyclin D1 (M-20), anti-cyclin E (HE-12), anti-cyclin A (BF-683), anti-CDK4 (H-22), and anti-CDK2 (M-2) (Santa Cruz Biotechnology, Santa Cruz, CA). As secondary antibodies, peroxidase-conjugated antimouse or antirabbit antibodies (Amersham) were used, followed by enhanced chemiluminescence (Amersham) detection. Some membranes were reused for new staining. The density of the bands was assessed using Multianalyst software.

Proliferation Assay.
Cells were cultured in 35-mm dishes. After the AS treatments, [3H]thymidine (1 µCi/ml, 5 Ci/mM) was added 1 h before termination of the experiments. The cells were rinsed with PBS, fixed with 10% (w/v) trichloroacetic acid, rinsed again with PBS, and finally dissolved in 1 ml of 1 M NaOH for scintillation counting and protein quantification. Assay of cell growth was performed using the Cell proliferation kit II (Roche Inc., Indianapolis, IN) as described elsewhere (23) . All standards and experiments were performed in triplicate.

Analysis of Cyclin D1 Synthesis and Degradation.
After the indicated experimental procedures, cells were transferred to methionine-free DMEM supplemented with 10% FBS and 100 µCi/ml L-[35S]methionine (specific activity >1000 Ci/mM; Amersham) for a 30-min incubation. For determination of cyclin D1 protein synthesis, the cells were quickly washed twice with ice-cold PBS and lysed in radioimmunoprecipitation assay buffer (radioimmunoprecipitation assay buffer, 1x PBS, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS, supplemented with protease inhibitor tablet). An equal amount of protein from each sample was immunoprecipitated with a polyclonal anti-cyclin D1 antibody (M-20), collected by protein A-Sepharose (CL-4B; Amersham), resolved by SDS-PAGE, and visualized by autoradiography.

Cyclin D1 protein degradation was determined by pulse-chase experiments. After the 30-min labeling with [35S]methionine (see above), the cells were carefully washed and transferred to radioactive-free DMEM containing 10% FBS for the indicated time periods. Cells were then harvested for detection of radioactive cyclin D1 as described above.

Cell Transfection.
Mouse fibroblast NIH3T3 cells were stably transfected with 2 µg pTargeT (Promega, Madison, WI) SYT-SSX2 plasmid DNA using a gene pulser apparatus, as described elsewhere (24) . To obtain stable transfected cells, 24 h after transfection the cells were treated with 800 µg/ml G418 (Promega) for 2 weeks, and then the drug-resistant colonies were maintained for use. The cells were analyzed regularly for SYT-SSX expression.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition of SYT-SSX Expression.
We designed three pairs of primers that were specific for SYT, SSX, or SYT-SSX transcripts (Fig. 1ACitation , left panel). Two synovial sarcoma cell lines, 1273/99 and CME-1, the Ewing’s sarcoma cell line RD-ES, and two melanoma cell lines, MEL-28 and DFW, were included in the experiments. After reverse transcription, semiquantitative PCR was performed. Actin was used as an internal control. All five cell lines expressed SYT. SSX was strongly expressed in RD-ES and in both melanoma cell lines, but SSX expression was very weak or absent in CME-1 and 1273/99, respectively. As expected, SYT-SSX was only detected in 1273/99 and CME-1 cells (Fig. 1ACitation , right panel).



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  		Fig. 1. Inhibition of SYT-SSX fusion gene expression using AS ODNs. A, the left panel shows the positions of the PCR primers used for analysis of wt SYT, wt SSX, and SYT-SSX expression (for details, see "Materials and Methods"). The right panel shows RT-PCR detection of wt SYT, wt SSX, and SYT-SSX transcripts in the CME-1, 1273/99, RD-ES, MEL-28, and DFW cell lines. B, positions of the four AS ODNs used to inhibit wt SYT, wt SSX, and SYT-SSX expression. C, 1273/99 cells either remained untreated (C) or were treated for 24 h with Lipofectin (L), Lipofectin + the indicated AS ODNs (AS), or Lipofectin + corresponding sense ODNs (S). Total RNA was isolated, and semiquantitative RT-PCR analysis for SYT-SSX and SYT mRNA was performed.

 
We designed four AS ODNs and corresponding sense ODNs as controls to inhibit SYT-SSX expression. The AS ODN binding sites are depicted schematically in Fig. 1BCitation . The four AS ODNs would interfere with SYT, SSX, and SYT-SSX expression as follows: AS1 inhibits both SYT and SYT-SSX expression; AS2 blocks SYT-SSX because it covers the breakpoint region of the fusion transcript; AS3 selectively interferes with SYT because the target region is not involved in the fusion transcript; and AS4 disrupts both SYT-SSX and SSX expression.

To date, there are no reliable antibodies available that specifically recognize the wt SYT and SSX proteins or the fusion protein created by the SYT-SSX translocation. We therefore evaluated the effects of AS ODNs on the targeted transcripts using semiquantitative RT-PCR. After 24-h treatments of 1273/99 cells with a type of AS ODNs (AS1-AS4) or corresponding sense ODNs, analysis of SYT-SSX and SYT transcripts were performed. We could confirm that AS1, AS2, and AS4 inhibited SYT-SSX, whereas SYT was blocked by AS1 and AS3 but not affected by AS2 and AS4 (Fig. 1C)Citation . This means that AS2 is specific for SYT-SSX and that AS3 is specific for SYT. In contrast, neither sense ODNs nor Lipofectin (used to increase the uptake of ODNs) had any effects on the targeted mRNAs. We also used mismatch AS ODNs (see "Materials and Methods") as negative controls in several experiments. Like sense ODNs, they did not alter the levels of the studied transcripts (data not shown). Our conclusion is that AS1-AS4 specifically inhibit the targeted transcripts.

Inhibition of SYT-SSX Down-Regulates Cyclin D1 and Reduces Cell Proliferation.
We investigated the effect of SYT-SSX inhibition induced by AS ODNs on major cell cycle proteins in synovial sarcoma cells. The 1273/99 cells were treated with AS1 and AS2, both of which blocked expression of the SYT-SSX fusion gene (Fig. 1C)Citation . After a 24-h incubation with AS ODN, the levels of cyclin D1, cyclin E, cyclin A, CDK4, and CDK2 were assessed by Western blotting. Actin was used as a loading control. The cyclin D1 expression was drastically decreased, whereas cyclin E and cyclin A were only moderately and slightly decreased, respectively. In contrast, CDK4 and CDK2 remained essentially unchanged (Fig. 2A)Citation . There were no differences between AS1 and AS2 (specific for SYT-SSX) with regard to the effect on cyclin D1. We also treated the 1273/99 cells with AS3 (specific for SYT) and AS4 (specific for SYT-SSX in the 1273/99 cells because they do not express SSX; see above). AS3 did not alter the cyclin D1 expression, whereas AS4, like AS1 and AS2, was efficient in this respect (Fig. 2B)Citation .



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  		Fig. 2. Effect of SYT-SSX inhibition on cell cycle proteins. A, 1273/99 cells were treated with AS1 or AS2 for 24 h, and then the cyclins and their corresponding CDKs were analyzed by Western blotting. B, 1273/99 cells were treated with AS3 or AS4 for 24 h, and then cyclin D1 was analyzed by Western blotting. C, two SYT-SSX-negative cell lines, RD-ES and DFW, were treated with the indicated AS ODNs for 24 h and analyzed for cyclin D1 expression.

 
The Ewing’s sarcoma cell line RD-ES and the melanoma cell line DFW, both of which express wt SYT and SSX but not the SYT-SSX fusion gene (Fig. 1A)Citation , were used as controls. We treated these cells with AS1 and AS4. Using semiquantitative RT-PCR, we confirmed that SYT and SSX mRNA expression was inhibited (data not shown). After AS1and AS4 treatments, we found that cyclin D1 expression was not inhibited in these cell lines (Fig. 2C)Citation . Taken together, the results strongly suggest that the inhibition of SYT-SSX specifically down-regulates cyclin D1. Because AS1, AS2, and AS4 each specifically down-regulated cyclin D1 in synovial sarcoma cells, we only used one or two AS ODNs (AS1 and AS2) in the following experiments.

To further confirm the results obtained on the 1273/99 cell line, we repeated the experiments on CME-1 cells. We could verify that AS1 or AS2 also blocked the expression of SYT-SSX mRNA in this synovial sarcoma cell line (Fig. 3A)Citation , and we found that the cyclin D1 protein almost completely disappeared (Fig. 3B)Citation . We also demonstrated that AS treatments reduced cell proliferation, as assayed by [3H]thymidine incorporation, in both CME-1 and 1273/99 cells (Fig. 3C)Citation . The growth curves of 1273/99 cells treated with AS1 and AS2 are shown in right panel of Fig. 3CCitation . It was confirmed that inhibition of SYT-SSX reduced proliferation of these cells. Furthermore, we analyzed the effects of SYT-SSX on proliferation of NIH3T3 and 3Y1 cells, which were transfected with the fusion gene. 3Y1 transfectants have been carefully characterized for SYT-SSX expression elsewhere (11) . In the left panel of Fig. 3DCitation , it is confirmed that NIH3T3/SYT-SSX cells express SYT-SSX. In the middle and right panels of Fig. 3DCitation , it is shown that the transfected cells exhibit a higher proliferative rate compared with wt cells and mock-transfected cells. This means that the fusion gene accelerates cell growth.



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  		Fig. 3. The effect of SYT-SSX inhibition on 1273/99 and CME-1 cell proliferation. CME-1 cells were treated with AS1 or AS2 and the corresponding sense ODNs for 24 h, and then the levels of SYT-SSX transcripts (A) and cyclin D1 protein (B) were determined by semiquantitative RT-PCR and Western blotting, respectively. C, CME-1 and 1273/99 cells were treated with AS1, AS2, or Lipofectin alone for 12–48 h, and then DNA synthesis was assayed by [3H]thymidine incorporation (left and middle panels). Cell growth of 1273/99 cells treated with AS1 and AS2 (right panel). D, detection of SYT-SSX transcript in transfected NIH3T3 cells (left panel). Growth of NIH3T3/SYT-SSX and 3Y1/SYT-SSX cells compared with wt cells and mock-transfected cells (middle and right panels), respectively.

 
Kinetics of Cyclin D1 Down-Regulation.
To investigate the kinetics of SYT-SSX inhibition of cyclin D1 expression, 1273/99 cells were treated with AS1 and AS2 for different time periods. After 3 h of AS treatment, the cyclin D1 protein level decreased about 75% (as measured by densitometry), and after 6 h, it was almost undetectable (Fig. 4)Citation . In contrast, the levels of CDK4 remained largely unchanged. These data indicate that the mechanisms underlying cyclin D1 down-regulation after SYT-SSX inhibition act fast.



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  		Fig. 4. Kinetics of cyclin D1 down-regulation. 1273/99 cells were exposed to AS1 or AS2 for the indicated times (3–24 h), Cyclin D1 and CDK4 proteins were analyzed by Western blotting.

 
Inhibition of SYT-SSX Destabilizes Cyclin D1.
The decrease in cyclin D1 protein could potentially be due to a decreased rate of synthesis or an accelerated rate of degradation. Therefore, we now investigated at what level SYT-SSX may affect the expression of cyclin D1 in synovial sarcoma cells, i.e., whether the fusion gene product interfered with transcription, protein synthesis, or ubiquitin-dependent degradation of cyclin D1. The 1273/99 cells were treated with AS1 or AS2 for 24 h, and then the level of cyclin D1 mRNA was analyzed by semiquantitative RT-PCR. Both SYT-SSX and cyclin D1 mRNA were detected using the same samples. Actin was used as an internal control. Whereas the fusion gene mRNA was dramatically decreased after treatment with AS, no decrease was observed for cyclin D1 mRNA expression (Fig. 5A)Citation . The 1273/99 cells were also treated with AS1 or AS2 for 3 or 12 h, respectively. Despite the fact that the SYT-SSX fusion gene was blocked at these time points, no cyclin D1 mRNA decrease was observed (data not shown). Thus, our conclusion is that inhibition of SYT-SSX does not affect cyclin D1 transcription.



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  		Fig. 5. Level of interference with cyclin D1. A, 1273/99 cells were treated with AS1 and AS2 for 24 h. Total RNA was isolated, and SYT-SSX and cyclin D1 mRNA were analyzed by semiquantitative RT-PCR. B, 1273/99 cells were treated with the proteasome inhibitor ALLN (50 µM) for 0–6 h. Cyclin D1 protein was detected by Western blotting. C, 1273/99 cells were treated with AS1 or AS2 with or without ALLN (50 µM) for 3 or 6 h as indicated. Cyclin D1 protein was determined by Western blotting.

 
Several studies have shown that G1 cyclins are largely regulated by their degradation via the ubiquitin-proteasome pathway (25 , 26) . For this reason, we analyzed whether SYT-SSX could interfere with degradation of cyclin D1 through this pathway. First, the cells were treated only with the proteasome inhibitor ALLN (50 µM) and harvested for assay of cyclin D1 expression after different incubation times (0–6 h). ALLN did not influence the levels of cyclin D1 protein (Fig. 5B)Citation . Next, the cells were treated with either AS1 or AS2 alone or together with ALLN for 3 and 6 h. As expected, the level of cyclin D1 was markedly reduced in cells treated with only AS as compared with untreated cells. However, in the presence of ALLN, the decrease in cyclin D1 protein was almost counteracted (Fig. 5C)Citation . These data implicated that cyclin D1 down-regulation due to inhibition of SYT-SSX expression could occur at the level of degradation through the ubiquitin-proteasome pathway.

In light of the above results, the 1273/99 cells were treated with AS1 at different time points and labeled with [35S]methionine for 30 min (see "Materials and Methods"). Using this assay, we could first investigate whether inhibition of SYT-SSX affects the synthesis of cyclin D1. Whereas 3- and 6-h treatments with AS ODN did not influence the synthesis of cyclin D1, a 16-h treatment resulted in an almost complete inhibition (Fig. 6A)Citation . In an independent experiment, we found that cyclin D1 protein synthesis started decreasing after an 8-h AS treatment and was dramatically decreased after 12 h (data not shown). Western blotting data clearly demonstrated that down-regulation of cyclin D1 was very rapid after AS treatment, e.g., after an AS treatment for 6 h, it was almost undetectable (see Fig. 4Citation ). Therefore, the lowered cyclin D1 protein synthesis cannot be the cause of the rapid cyclin D1 decrease. To address this question further, 1273/99 cells were treated with AS1 for 6 h (as shown in Fig. 6ACitation , cyclin D1 synthesis was not affected at this time point) and then subjected to pulse-chase labeling with [35S]methionine. As shown in Fig. 6BCitation , cyclin D1 of AS ODN-treated cells is less stable. Using densitometry, it was determined that the t1/2 (half-life) of cyclin D1 protein was 35 ± 4 min, compared with 20 ± 2 min in SYT-SSX-inhibited cells (three independent experiments). These data suggest that cyclin D1 protein degradation is the mechanism for the decrease in cyclin D1 expression in SYT-SSX-blocked cells.



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  		Fig. 6. Cyclin D1 protein synthesis and degradation. A, cyclin D1 protein synthesis. 1273/99 cells were treated with AS1 for 3, 6, or 16 h, and then the cells were labeled with [35S]methionine (100 µCi/ml) for 30 min. Cyclin D1 was immunoprecipitated, resolved by SDS-PAGE, and finally visualized by autoradiography. B, degradation of cyclin D1 protein. 1273/99 cells were treated with AS1 for 6 h, labeled with [35S]methionine (100 µCi/ml) for 30 min, and then chased for the indicated times. Cyclin D1 was immunoprecipitated, resolved by SDS-PAGE, and visualized by autoradiography. C, the degradation rate of cyclin D1 in NIH3T3 and NIH3T3/SYT-SSX cells was determined by pulse-chase experiments, as described above. D, effect of SYT-SSX inhibition on cyclin D1 expression in NIH3T3/SYT-SSX cells. The cells were treated with AS1 for 24 h, and then cyclin D1 was analyzed by Western blotting.

 
Furthermore, we analyzed whether SYT-SSX could affect cyclin D1 half-life in NIH3T3 cells. The wt and stable SYT-SSX-transfected NIH3T3 cells (NIH3T3/SYT-SSX) were labeled with [35S]methionine for 30 min and chased for the indicated times. The t1/2 of the cyclin D1 protein in NIH3T3 cells was 25 ± 3 min, compared with 40 ± 5 min in NIH3T3/SYT-SSX cells (three independent experiments). Thus, the half-life was substantially extended due to SYT-SSX expression in NIH3T3 cells (Fig. 6C)Citation . Consistently, it was confirmed that SYT-SSX inhibition decreased cyclin D1 expression in these cells (Fig. 6D)Citation .

Cyclin D1 Degradation Is Independent of the PI3K-Akt-GSK-3ß Pathway.
Recent studies have shown that the PI3K-Akt-GSK-3ß pathway is involved in ubiquitin-proteasome-dependent degradation of cyclin D1 in certain cell types (27) . This prompted us to investigate whether SYT-SSX could interfere with this mechanism in synovial sarcoma cells. PI3K is known to phosphorylate Akt to pAkt, which in turn phosphorylates GSK-3ß to pGSK-3ß (25) . Unphosphorylated GSK-3ß, being the active isoform, phosphorylates cyclin D1 on threonine 286 and triggers cyclin D1 degradation via the ubiquitin-dependent pathway (27 , 28) . Consequently, mitogenic stimuli, which activate the Ras-PI3K-Akt pathway, lead to decreased levels of GSK-3ß and increased cyclin D1 expression (27) .

LY294002, a PI3K inhibitor, can specifically block the PI3K-Akt-GSK-3ß pathway (29) . 1273/99 cells were treated with LY294002 (30 µM) for indicated times. As clearly demonstrated, cyclin D1 protein was drastically decreased by treatment with LY294002 for 3 h and was almost undetectable at 6 h. This response was paralleled by a decrease in pAkt and pGSK-3ß, whereas total Akt and GSK-3ß remained unchanged (Fig. 7ACitation , left panel). Similar results were observed in CME-1, NIH3T3, and NIH3T3/SYT-SSX cells (data not shown). This result confirms that the PI3K-Akt-GSK-3ß pathway is a common mechanism in cyclin D1 regulation and also exists in synovial sarcoma cells.



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  		Fig. 7. Analysis of the PI3K-Akt-GSK-3ß pathway. A, 1273/99 cells were treated with LY294002 (30 µM, left panel) or AS1 (right panel) for 3, 6, and 9 h. The cell proteins were extracted, and Western blotting was performed to detect cyclin D1, Akt, GSK-3ß, and the phosphorylated products pAkt and pGSK-3ß. B, 1273/99 cells were treated with AS1 with or without LiCl (20 mM) for 3 or 6 h. Cyclin D1 protein was analyzed by Western blotting.

 
Based on the above-mentioned experiment, we now investigated whether SYT-SSX regulates cyclin D1 stability by interfering with the PI3K-Akt-GSK-3ß pathway. 1273/99 cells were treated with AS1 for 3 and 6 h. In contrast to cyclin D1, which was expectedly decreased, the expression of total GSK-3ß and Akt as well as their phosphorylated forms remained completely unchanged after SYT-SSX inhibition (Fig. 7ACitation , right panel).

To further confirm these results, we treated the 1273/99 cells with lithium chloride (LiCl), which inactivates the kinase activity of GSK-3ß (30 , 31) . However, LiCl (20 mM) did not counteract the decrease in cyclin D1 after treatment with AS (Fig. 7B)Citation . Our data therefore suggest that SYT-SSX-dependent cyclin D1 protein regulation in synovial sarcoma cells is independent of the PI3K-Akt-GSK-3ß pathway.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The chromosomal translocation t(X;18)(p11.2;q11.2) is the key genetic event in synovial sarcoma, and it is becoming increasingly evident that SYT-SSX plays a crucial role in tumor progression and development (1 , 32) . However, the underlying molecular mechanisms are still largely unknown.

In this study, we found that inhibition of SYT-SSX induces a rapid and selective decrease in cyclin D1, followed by a decline in proliferation, suggesting that SYT-SSX is critical for stable expression of cyclin D1 and growth of synovial sarcoma cells. Because it is well established that a high expression of cyclin D1 is important for tumor cell proliferation and oncogenesis (19 , 33) , our data provide evidence that SYT-SSX-dependent cyclin D1 expression may be a critical event in the development and progression of synovial sarcoma.

There was also a slight to moderate decrease in cyclin E expression in SYT-SSX-inhibited cells, but because cyclin E is a major downstream target of cyclin D1 (34) , we believe that this event may be secondary to the depletion of cyclin D1.

Another significant finding is that the SYT-SSX-dependent expression of cyclin D1 is specific for the fusion gene. We could conclude that the wt genes SYT and SSX are not involved. This result is consistent with the study by Nagai et al. (11) showing that SYT or SSX, in contrast to SYT-SSX, could not enhance cell proliferation and had no transforming activity. The parts of SYT and SSX involved in the fusion protein contain transcriptional activating regions and repressor domains, respectively (8) . Perhaps the conformational changes after the fusion between SYT and SSX are important for the function of the fusion protein, thereby acquiring new properties that are not present in the wt proteins. For example, due to conformational changes, the transactivating ability of SYT might be augmented, or the interactions with other proteins could be altered. The recent finding that SYT-SSX is functionally linked to the transcriptional regulatory machinery involving hBRM/hSNF2a (11) suggests that suppression and activation of target genes may play a central role in directing the expression of proteins crucial for the development of synovial sarcoma. One or more such target genes might be involved in stabilization or destabilization of cyclin D1.

Our results demonstrate that SYT-SSX regulates cyclin D1 expression at the degradation level. Regulation of cyclin D1 may otherwise occur at the transcriptional and translational level (18 , 35 , 36) , but rapid and drastic alterations in cyclin D1 levels are usually modulated via protein degradation (25 , 28 , 37) . This is explained by the fact that cyclin D1 is a very labile protein. In this study, we show that a proteasome inhibitor efficiently prevented the decrease in cyclin D1 expression after inhibition of SYT-SSX, suggesting that the SYT-SSX protein interferes with cyclin D1 expression via the ubiquitin-proteasome pathway. Experiments using pulse-chase labeling with [35S] methionine confirmed that SYT-SSX inhibition increased degradation of cyclin D1. However, the action of GSK-3ß does not seem to be involved in this processing in synovial sarcoma cells. This suggests that other mechanisms may be involved in degradation of cyclin D1 due to SYT-SSX inhibition. Even in other cell systems, it has been demonstrated that cyclin D1 degradation could be independent of GSK-3ß (25) . Recently, a destruction motif (destruction box) was identified in cyclin D1 (37) . This destruction box, independent of the PI3K-Akt-GSK-3ß pathway, is necessary for rapid cyclin D1 degradation due to genotoxic stress (37) . These studies point to the existence of alternative pathways, still unknown, involved in ubiquitin-proteasome-dependent degradation of cyclin D1. Because SYT-SSX expression markedly stabilized cyclin D1 in transfected NIH3T3 cells, it appears as the SYT-SSX protein interferes (directly or indirectly) with a mechanism that is common for mammalian cells and not specific for synovial sarcoma.

Although the rapid cyclin D1 decrease (75% decrease after 3 h), obtained by SYT-SSX inhibition seems to be due to an accelerated proteolysis, we also observed that cyclin D1 protein synthesis was affected. However, this was a comparatively slow event, and during the first 6 h of AS ODN treatment, no detectable inhibitory effect on cyclin D1 protein synthesis was observed. Because the decrease in cyclin D1 synthesis occurs so late, it cannot be excluded that this event is secondary, e.g., to the ongoing growth retardation.

In contrast, we could not detect any effects of SYT-SSX inhibition on cyclin D1 at the mRNA level, at least not during the first 24 h. However, this is not so surprising, because it has also been demonstrated in other experimental systems that down-regulation of the cyclin D1 protein does not correlate well with cyclin D1 mRNA expression (25 , 37 , 38) .

The role of SYT-SSX in expression of cyclin D1, as demonstrated in this study, may be important for transformation and progression of synovial sarcoma and may have therapeutic implications because inhibition of SYT-SSX could selectively suppress growth of synovial sarcoma cells while sparing other tissues.


   ACKNOWLEDGMENTS
 
We thank Patricia K. Stoltzfus for language revision of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Swedish Cancer Society, the Cancer Society in Stockholm, the Swedish Children Cancer Society, and the Karolinska Institute. A. G. was supported by a fellowship from the Fondazione Italiana per la Ricerca sul Cancro. Back

2 To whom requests for reprints should be addressed, at CCK R8:04, Department of Oncology and Pathology, Karolinska Hospital, SE-171 76 Stockholm, Sweden. Fax: 46-8-7588397; E-mail: olle.larsson{at}onkpat.ki.se Back

3 The abbreviations used are: AS, antisense; ODN, oligonucleotide; ALLN, N-acetyl-Leu-Leu-Norleucinal; wt, wild-type; PI3K, phosphatidylinositol 3'-kinase; Akt, protein kinase B; GSK, glycogen synthetase kinase; FBS, fetal bovine serum; RT-PCR, reverse transcription-PCR; CDK, cyclin-dependent kinase. Back

Received 2/ 4/02. Accepted 4/25/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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