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Inhibition of constitutively active Jak-Stat pathway suppresses cell growth of human T-cell leukemia virus type 1-infected T-cell lines and primary adult T-cell leukemia cells.

Tomita M, Kawakami H, Uchihara JN, Okudaira T, Masuda M, Matsuda T, Tanaka Y, Ohshiro K, Mori N - Retrovirology (2006)

Bottom Line: Using AG490, a Jak-specific inhibitor, we demonstrated that the activation of Stat3 and Stat5 was mediated by the constitutive phosphorylation of Jak proteins.AG490 inhibited the growth of HTLV-1-infected T-cell lines and primary ATL cells by inducing G1 cell-cycle arrest mediated by altering the expression of cyclin D2, Cdk4, p53, p21, Pim-1 and c-Myc, and by apoptosis mediated by the reduced expression of c-IAP2, XIAP, survivin and Bcl-2.Inhibition of this pathway may provide a new approach for the treatment of ATL.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular Virology and Oncology, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan. mtomita@med.u-ryukyu.ac.jp

ABSTRACT

Background: Human T-cell leukemia virus type 1 (HTLV-1), the etiologic agent for adult T-cell leukemia (ATL), induces cytokine-independent proliferation of T-cells, associated with the acquisition of constitutive activation of Janus kinases (Jak) and signal transducers and activators of transcription (Stat) proteins. Our purposes in this study were to determine whether activation of Jak-Stat pathway is responsible for the proliferation and survival of ATL cells, and to explore mechanisms by which inhibition of Jak-Stat pathway kills ATL cells.

Results: Constitutive activation of Stat3 and Stat5 was observed in HTLV-1-infected T-cell lines and primary ATL cells, but not in HTLV-1-negative T-cell lines. Using AG490, a Jak-specific inhibitor, we demonstrated that the activation of Stat3 and Stat5 was mediated by the constitutive phosphorylation of Jak proteins. AG490 inhibited the growth of HTLV-1-infected T-cell lines and primary ATL cells by inducing G1 cell-cycle arrest mediated by altering the expression of cyclin D2, Cdk4, p53, p21, Pim-1 and c-Myc, and by apoptosis mediated by the reduced expression of c-IAP2, XIAP, survivin and Bcl-2. Importantly, AG490 did not inhibit the growth of normal peripheral blood mononuclear cells.

Conclusion: Our results indicate that activation of Jak-Stat pathway is responsible for the proliferation and survival of ATL cells. Inhibition of this pathway may provide a new approach for the treatment of ATL.

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Effects of AG490 on the expression of anti-apoptotic proteins. (A) HTLV-1-infected T-cell lines were treated with increasing concentrations of AG490 for 24 h. Amounts of c-IAP-2, XIAP, survivin, Bcl-2, Bcl-xL and Tax were determined by Western blot analysis. (B) Primary ATL cells were treated with (+) or without (-) 50 μM AG490 for 24 h. The expression of c-IAP2, XIAP, survivin and Tax was assessed by Western blot analysis. (C) HUT-102 cells were treated with (+) or without (-) 50 μM AG490 for 24 h. The expression of HTLV-1 viral proteins, envelope glycoprotein gp46 and p19 core protein was assessed by Western blot analysis. The amount of actin is shown as a loading control.
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Figure 6: Effects of AG490 on the expression of anti-apoptotic proteins. (A) HTLV-1-infected T-cell lines were treated with increasing concentrations of AG490 for 24 h. Amounts of c-IAP-2, XIAP, survivin, Bcl-2, Bcl-xL and Tax were determined by Western blot analysis. (B) Primary ATL cells were treated with (+) or without (-) 50 μM AG490 for 24 h. The expression of c-IAP2, XIAP, survivin and Tax was assessed by Western blot analysis. (C) HUT-102 cells were treated with (+) or without (-) 50 μM AG490 for 24 h. The expression of HTLV-1 viral proteins, envelope glycoprotein gp46 and p19 core protein was assessed by Western blot analysis. The amount of actin is shown as a loading control.

Mentions: We also examined the effects of AG490 on the expression of IAP and Bcl-2 family members, which determine the response to apoptotic stimuli. AG490 significantly altered the expression of XIAP and survivin, which are Stat-regulated genes [25,26], but not that of Bcl-xL protein in all tested cell lines (Figure 6A). Downregulation of Bcl-2 expression by AG490 was only noted in HUT-102 cells. The expression of c-IAP2 was downregulated in HUT-102 and ED-40515(-), but not in MT-2 cells. These results indicated that AG490-induced apoptosis of HTLV-1-infected T-cells is mediated by downregulation of c-IAP2, XIAP, survivin and Bcl-2 expression. AG490 reduced the expression of all these genes in freshly isolated ATL cells (Figure 6B). Bcl-2 protein was undetectable in primary ATL cells (data not shown). Cyclin D2 [27,28], Cdk4 [29], XIAP [30] and survivin [31] are Tax-responsive genes, therefore, we also examined the level of Tax expression in these cells. AG490 did not alter Tax protein levels in MT-2 and HUT-102 cells (Figure 6A). Tax protein remained at undetectable levels in ED-40515(-) and primary ATL cells after AG490 treatment (Figures 6A and 6B). Therefore, the altered expression of cyclin D2, Cdk4, XIAP and survivin was not attributable to Tax downregulation. We also examined whether AG490 could change the expression levels of other viral proteins. The expression levels of HTLV-1 envelope 46 kDa glycoprotein (gp46) and 19 kDa core protein (p19) were not changed by AG490 treatment in HUT-102 cells (Figure 6C), suggesting that the AG490 does not drop the virus levels in these cells and the effects of AG490 on these cells are not due to downregulation of viral proteins.


Inhibition of constitutively active Jak-Stat pathway suppresses cell growth of human T-cell leukemia virus type 1-infected T-cell lines and primary adult T-cell leukemia cells.

Tomita M, Kawakami H, Uchihara JN, Okudaira T, Masuda M, Matsuda T, Tanaka Y, Ohshiro K, Mori N - Retrovirology (2006)

Effects of AG490 on the expression of anti-apoptotic proteins. (A) HTLV-1-infected T-cell lines were treated with increasing concentrations of AG490 for 24 h. Amounts of c-IAP-2, XIAP, survivin, Bcl-2, Bcl-xL and Tax were determined by Western blot analysis. (B) Primary ATL cells were treated with (+) or without (-) 50 μM AG490 for 24 h. The expression of c-IAP2, XIAP, survivin and Tax was assessed by Western blot analysis. (C) HUT-102 cells were treated with (+) or without (-) 50 μM AG490 for 24 h. The expression of HTLV-1 viral proteins, envelope glycoprotein gp46 and p19 core protein was assessed by Western blot analysis. The amount of actin is shown as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1483830&req=5

Figure 6: Effects of AG490 on the expression of anti-apoptotic proteins. (A) HTLV-1-infected T-cell lines were treated with increasing concentrations of AG490 for 24 h. Amounts of c-IAP-2, XIAP, survivin, Bcl-2, Bcl-xL and Tax were determined by Western blot analysis. (B) Primary ATL cells were treated with (+) or without (-) 50 μM AG490 for 24 h. The expression of c-IAP2, XIAP, survivin and Tax was assessed by Western blot analysis. (C) HUT-102 cells were treated with (+) or without (-) 50 μM AG490 for 24 h. The expression of HTLV-1 viral proteins, envelope glycoprotein gp46 and p19 core protein was assessed by Western blot analysis. The amount of actin is shown as a loading control.
Mentions: We also examined the effects of AG490 on the expression of IAP and Bcl-2 family members, which determine the response to apoptotic stimuli. AG490 significantly altered the expression of XIAP and survivin, which are Stat-regulated genes [25,26], but not that of Bcl-xL protein in all tested cell lines (Figure 6A). Downregulation of Bcl-2 expression by AG490 was only noted in HUT-102 cells. The expression of c-IAP2 was downregulated in HUT-102 and ED-40515(-), but not in MT-2 cells. These results indicated that AG490-induced apoptosis of HTLV-1-infected T-cells is mediated by downregulation of c-IAP2, XIAP, survivin and Bcl-2 expression. AG490 reduced the expression of all these genes in freshly isolated ATL cells (Figure 6B). Bcl-2 protein was undetectable in primary ATL cells (data not shown). Cyclin D2 [27,28], Cdk4 [29], XIAP [30] and survivin [31] are Tax-responsive genes, therefore, we also examined the level of Tax expression in these cells. AG490 did not alter Tax protein levels in MT-2 and HUT-102 cells (Figure 6A). Tax protein remained at undetectable levels in ED-40515(-) and primary ATL cells after AG490 treatment (Figures 6A and 6B). Therefore, the altered expression of cyclin D2, Cdk4, XIAP and survivin was not attributable to Tax downregulation. We also examined whether AG490 could change the expression levels of other viral proteins. The expression levels of HTLV-1 envelope 46 kDa glycoprotein (gp46) and 19 kDa core protein (p19) were not changed by AG490 treatment in HUT-102 cells (Figure 6C), suggesting that the AG490 does not drop the virus levels in these cells and the effects of AG490 on these cells are not due to downregulation of viral proteins.

Bottom Line: Using AG490, a Jak-specific inhibitor, we demonstrated that the activation of Stat3 and Stat5 was mediated by the constitutive phosphorylation of Jak proteins.AG490 inhibited the growth of HTLV-1-infected T-cell lines and primary ATL cells by inducing G1 cell-cycle arrest mediated by altering the expression of cyclin D2, Cdk4, p53, p21, Pim-1 and c-Myc, and by apoptosis mediated by the reduced expression of c-IAP2, XIAP, survivin and Bcl-2.Inhibition of this pathway may provide a new approach for the treatment of ATL.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular Virology and Oncology, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan. mtomita@med.u-ryukyu.ac.jp

ABSTRACT

Background: Human T-cell leukemia virus type 1 (HTLV-1), the etiologic agent for adult T-cell leukemia (ATL), induces cytokine-independent proliferation of T-cells, associated with the acquisition of constitutive activation of Janus kinases (Jak) and signal transducers and activators of transcription (Stat) proteins. Our purposes in this study were to determine whether activation of Jak-Stat pathway is responsible for the proliferation and survival of ATL cells, and to explore mechanisms by which inhibition of Jak-Stat pathway kills ATL cells.

Results: Constitutive activation of Stat3 and Stat5 was observed in HTLV-1-infected T-cell lines and primary ATL cells, but not in HTLV-1-negative T-cell lines. Using AG490, a Jak-specific inhibitor, we demonstrated that the activation of Stat3 and Stat5 was mediated by the constitutive phosphorylation of Jak proteins. AG490 inhibited the growth of HTLV-1-infected T-cell lines and primary ATL cells by inducing G1 cell-cycle arrest mediated by altering the expression of cyclin D2, Cdk4, p53, p21, Pim-1 and c-Myc, and by apoptosis mediated by the reduced expression of c-IAP2, XIAP, survivin and Bcl-2. Importantly, AG490 did not inhibit the growth of normal peripheral blood mononuclear cells.

Conclusion: Our results indicate that activation of Jak-Stat pathway is responsible for the proliferation and survival of ATL cells. Inhibition of this pathway may provide a new approach for the treatment of ATL.

Show MeSH
Related in: MedlinePlus