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ASPL-TFE3 Oncoprotein Regulates Cell Cycle Progression and Induces Cellular Senescence by Up-Regulating p21

View Article: PubMed Central - PubMed

ABSTRACT

Alveolar soft part sarcoma is an extremely rare soft tissue sarcoma with poor prognosis. It is characterized by the unbalanced recurrent chromosomal translocation der(17)t(X;17)(p11;q25), resulting in the generation of an ASPL-TFE3 fusion gene. ASPL-TFE3 oncoprotein functions as an aberrant transcriptional factor and is considered to play a crucial role in the tumorigenesis of alveolar soft part sarcoma. However, the underlying molecular mechanisms are poorly understood. In this study, we identified p21 (p21WAF1/CIP1) as a direct transcriptional target of ASPL-TFE3. Ectopic ASPL-TFE3 expression in 293 cells resulted in cell cycle arrest and significant increases in protein and mRNA levels of p21. ASPL-TFE3 activated p21 expression in a p53-independent manner through direct transcriptional interactions with the p21 promoter region. When ASPL-TFE3 was expressed in human bone marrow–derived mesenchymal stem cells in a tetracycline-inducible manner, we observed the up-regulation of p21 expression and the induction of senescence-associated β-galactosidase activity. Suppression of p21 significantly decreased the induction of ASPL-TFE3-mediated cellular senescence. Furthermore, ASPL-TFE3 expression in mesenchymal stem cells resulted in a significant up-regulation of proinflammatory cytokines associated with senescence-associated secretory phenotype (SASP). These results show that ASPL-TFE3 regulates cell cycle progression and induces cellular senescence by up-regulating p21 expression. In addition, our data suggest a potential mechanism by which ASPL-TFE3-induced senescence may play a role in tumorigenesis by inducing SASP, which could promote the protumorigenic microenvironment.

No MeSH data available.


Related in: MedlinePlus

Effects of ASPL-TFE3 on cell proliferation and cell cycle progression. (A) 293/TR-AT cells were cultured in the presence of tetracycline for the indicated times and were then subjected to immunoblot analyses using the indicated antibodies. The expression levels of ASPL-TFE3 were examined using immunoblotting with anti-FLAG antibody. (B) 293/TR-AT cells were cultured in the presence or absence of tetracycline (+ or − Tet) for the indicated times and the cell numbers were determined. Experiments were performed twice with triplicate determinations for each time point. Data are presented as mean ± standard deviation (SD). (C) Cell cycle distributions of 293/TR-AT cells were analyzed using FACS. 293/TR-AT cells were cultured in the presence or absence of tetracycline (+ or − Tet) for the indicated times and were then stained with PI for FACS analyses.
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f0005: Effects of ASPL-TFE3 on cell proliferation and cell cycle progression. (A) 293/TR-AT cells were cultured in the presence of tetracycline for the indicated times and were then subjected to immunoblot analyses using the indicated antibodies. The expression levels of ASPL-TFE3 were examined using immunoblotting with anti-FLAG antibody. (B) 293/TR-AT cells were cultured in the presence or absence of tetracycline (+ or − Tet) for the indicated times and the cell numbers were determined. Experiments were performed twice with triplicate determinations for each time point. Data are presented as mean ± standard deviation (SD). (C) Cell cycle distributions of 293/TR-AT cells were analyzed using FACS. 293/TR-AT cells were cultured in the presence or absence of tetracycline (+ or − Tet) for the indicated times and were then stained with PI for FACS analyses.

Mentions: We established 293 cells that express ASPL-TFE3 in a tetracycline-inducible manner, and designated them as 293/TR-AT cells. Induction of ASPL-TFE3 expression by tetracycline addition was confirmed by immunoblotting (Figure 1A). To determine whether ASPL-TFE3 expression affects cell proliferation, 293/TR-AT cells were cultured with tetracycline and their proliferation was evaluated. As shown in Figure. 1B, the induction of ASPL-TFE3 expression in 293/TR-AT cells inhibited cell proliferation. Fluorescence-activated cell sorting (FACS) analysis revealed that ASPL-TFE3 expression resulted in an increase in the population of cells in the G0/G1 phase, with a concomitant decrease in the number of cells in the S phase (Figure 1C), suggesting that ASPL-TFE3 induces growth arrest in 293 cells.


ASPL-TFE3 Oncoprotein Regulates Cell Cycle Progression and Induces Cellular Senescence by Up-Regulating p21
Effects of ASPL-TFE3 on cell proliferation and cell cycle progression. (A) 293/TR-AT cells were cultured in the presence of tetracycline for the indicated times and were then subjected to immunoblot analyses using the indicated antibodies. The expression levels of ASPL-TFE3 were examined using immunoblotting with anti-FLAG antibody. (B) 293/TR-AT cells were cultured in the presence or absence of tetracycline (+ or − Tet) for the indicated times and the cell numbers were determined. Experiments were performed twice with triplicate determinations for each time point. Data are presented as mean ± standard deviation (SD). (C) Cell cycle distributions of 293/TR-AT cells were analyzed using FACS. 293/TR-AT cells were cultured in the presence or absence of tetracycline (+ or − Tet) for the indicated times and were then stained with PI for FACS analyses.
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Related In: Results  -  Collection

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f0005: Effects of ASPL-TFE3 on cell proliferation and cell cycle progression. (A) 293/TR-AT cells were cultured in the presence of tetracycline for the indicated times and were then subjected to immunoblot analyses using the indicated antibodies. The expression levels of ASPL-TFE3 were examined using immunoblotting with anti-FLAG antibody. (B) 293/TR-AT cells were cultured in the presence or absence of tetracycline (+ or − Tet) for the indicated times and the cell numbers were determined. Experiments were performed twice with triplicate determinations for each time point. Data are presented as mean ± standard deviation (SD). (C) Cell cycle distributions of 293/TR-AT cells were analyzed using FACS. 293/TR-AT cells were cultured in the presence or absence of tetracycline (+ or − Tet) for the indicated times and were then stained with PI for FACS analyses.
Mentions: We established 293 cells that express ASPL-TFE3 in a tetracycline-inducible manner, and designated them as 293/TR-AT cells. Induction of ASPL-TFE3 expression by tetracycline addition was confirmed by immunoblotting (Figure 1A). To determine whether ASPL-TFE3 expression affects cell proliferation, 293/TR-AT cells were cultured with tetracycline and their proliferation was evaluated. As shown in Figure. 1B, the induction of ASPL-TFE3 expression in 293/TR-AT cells inhibited cell proliferation. Fluorescence-activated cell sorting (FACS) analysis revealed that ASPL-TFE3 expression resulted in an increase in the population of cells in the G0/G1 phase, with a concomitant decrease in the number of cells in the S phase (Figure 1C), suggesting that ASPL-TFE3 induces growth arrest in 293 cells.

View Article: PubMed Central - PubMed

ABSTRACT

Alveolar soft part sarcoma is an extremely rare soft tissue sarcoma with poor prognosis. It is characterized by the unbalanced recurrent chromosomal translocation der(17)t(X;17)(p11;q25), resulting in the generation of an ASPL-TFE3 fusion gene. ASPL-TFE3 oncoprotein functions as an aberrant transcriptional factor and is considered to play a crucial role in the tumorigenesis of alveolar soft part sarcoma. However, the underlying molecular mechanisms are poorly understood. In this study, we identified p21 (p21WAF1/CIP1) as a direct transcriptional target of ASPL-TFE3. Ectopic ASPL-TFE3 expression in 293 cells resulted in cell cycle arrest and significant increases in protein and mRNA levels of p21. ASPL-TFE3 activated p21 expression in a p53-independent manner through direct transcriptional interactions with the p21 promoter region. When ASPL-TFE3 was expressed in human bone marrow–derived mesenchymal stem cells in a tetracycline-inducible manner, we observed the up-regulation of p21 expression and the induction of senescence-associated β-galactosidase activity. Suppression of p21 significantly decreased the induction of ASPL-TFE3-mediated cellular senescence. Furthermore, ASPL-TFE3 expression in mesenchymal stem cells resulted in a significant up-regulation of proinflammatory cytokines associated with senescence-associated secretory phenotype (SASP). These results show that ASPL-TFE3 regulates cell cycle progression and induces cellular senescence by up-regulating p21 expression. In addition, our data suggest a potential mechanism by which ASPL-TFE3-induced senescence may play a role in tumorigenesis by inducing SASP, which could promote the protumorigenic microenvironment.

No MeSH data available.


Related in: MedlinePlus