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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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AG490 reduces cell growth of HTLV-1-infected T-cell lines and primary ATL cells. (A) HTLV-1-infected T-cell lines (5 × 104/mL) were treated with 0, 25 or 50 μM AG490 for 24 or 48 h. Cell numbers were counted in triplicate by Trypan blue dye exclusion method. Data are expressed as the mean values of viable cell numbers. (B) Primary ATL cells from seven patients (ATL #1–7) and PBMCs from three healthy donors (Normal #1–3) were treated with 0, 25 or 50 μM AG490 for 48 h. Cell growth was assessed by the WST-8 method. Data are expressed as the percentages of control (untreated cells). (C) Cell-cycle analysis of HTLV-1-infected T-cell lines treated with AG490. Cells were treated in the absence (-) or presence (+) of 25 μM AG490 for 24 h. DNA content was analyzed by flow cytometry with propidium iodide staining. G1, S and G2/M indicate the stages of the cell-cycle. Data represent mean percentages of cells at each cell-cycle from three independent experiments. (D) Induction of apoptosis in HTLV-1-infected T-cell lines by AG490. Cells were treated in the absence (open bar) or presence (solid bar) of 50 μM AG490 for 48 h and stained with Annexin-V. Apoptosis was analyzed by flow cytometry. Data represent mean percentages of apoptotic cells from three independent experiments.
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Figure 4: AG490 reduces cell growth of HTLV-1-infected T-cell lines and primary ATL cells. (A) HTLV-1-infected T-cell lines (5 × 104/mL) were treated with 0, 25 or 50 μM AG490 for 24 or 48 h. Cell numbers were counted in triplicate by Trypan blue dye exclusion method. Data are expressed as the mean values of viable cell numbers. (B) Primary ATL cells from seven patients (ATL #1–7) and PBMCs from three healthy donors (Normal #1–3) were treated with 0, 25 or 50 μM AG490 for 48 h. Cell growth was assessed by the WST-8 method. Data are expressed as the percentages of control (untreated cells). (C) Cell-cycle analysis of HTLV-1-infected T-cell lines treated with AG490. Cells were treated in the absence (-) or presence (+) of 25 μM AG490 for 24 h. DNA content was analyzed by flow cytometry with propidium iodide staining. G1, S and G2/M indicate the stages of the cell-cycle. Data represent mean percentages of cells at each cell-cycle from three independent experiments. (D) Induction of apoptosis in HTLV-1-infected T-cell lines by AG490. Cells were treated in the absence (open bar) or presence (solid bar) of 50 μM AG490 for 48 h and stained with Annexin-V. Apoptosis was analyzed by flow cytometry. Data represent mean percentages of apoptotic cells from three independent experiments.

Mentions: Next we examined the effect of AG490 on the growth of HTLV-1-infected T-cell lines and primary ATL cells. HTLV-1-infected T-cell lines were treated with different concentration of AG490 (0, 25 or 50 μM) and cell numbers were counted 24 and 48 h after treatment. AG490 suppressed the growth of HTLV-1-infected T-cell lines in a dose and time dependent manner (Figure 4A). The antiproliferative effects of AG490 against primary ATL cells and PBMCs from healthy donors were measured by WST-8 method (Cell Counting Kit-8; Wako Chemical, Osaka, Japan) based on the MTT assay as described previously [20]. Cell viability was determined as percentage of the control (without AG490). AG490 also inhibited the growth of PBMCs from ATL patients (ATL #1–7 in Figure 4B). In comparison, the cell growth inhibitory effect on PBMCs from healthy donors was weak (Normal #1–3 in Figure 4B). These findings indicate that AG490 inhibits the growth of cells infected with HTLV-1 but not that of uninfected PBMCs.


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)

AG490 reduces cell growth of HTLV-1-infected T-cell lines and primary ATL cells. (A) HTLV-1-infected T-cell lines (5 × 104/mL) were treated with 0, 25 or 50 μM AG490 for 24 or 48 h. Cell numbers were counted in triplicate by Trypan blue dye exclusion method. Data are expressed as the mean values of viable cell numbers. (B) Primary ATL cells from seven patients (ATL #1–7) and PBMCs from three healthy donors (Normal #1–3) were treated with 0, 25 or 50 μM AG490 for 48 h. Cell growth was assessed by the WST-8 method. Data are expressed as the percentages of control (untreated cells). (C) Cell-cycle analysis of HTLV-1-infected T-cell lines treated with AG490. Cells were treated in the absence (-) or presence (+) of 25 μM AG490 for 24 h. DNA content was analyzed by flow cytometry with propidium iodide staining. G1, S and G2/M indicate the stages of the cell-cycle. Data represent mean percentages of cells at each cell-cycle from three independent experiments. (D) Induction of apoptosis in HTLV-1-infected T-cell lines by AG490. Cells were treated in the absence (open bar) or presence (solid bar) of 50 μM AG490 for 48 h and stained with Annexin-V. Apoptosis was analyzed by flow cytometry. Data represent mean percentages of apoptotic cells from three independent experiments.
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Figure 4: AG490 reduces cell growth of HTLV-1-infected T-cell lines and primary ATL cells. (A) HTLV-1-infected T-cell lines (5 × 104/mL) were treated with 0, 25 or 50 μM AG490 for 24 or 48 h. Cell numbers were counted in triplicate by Trypan blue dye exclusion method. Data are expressed as the mean values of viable cell numbers. (B) Primary ATL cells from seven patients (ATL #1–7) and PBMCs from three healthy donors (Normal #1–3) were treated with 0, 25 or 50 μM AG490 for 48 h. Cell growth was assessed by the WST-8 method. Data are expressed as the percentages of control (untreated cells). (C) Cell-cycle analysis of HTLV-1-infected T-cell lines treated with AG490. Cells were treated in the absence (-) or presence (+) of 25 μM AG490 for 24 h. DNA content was analyzed by flow cytometry with propidium iodide staining. G1, S and G2/M indicate the stages of the cell-cycle. Data represent mean percentages of cells at each cell-cycle from three independent experiments. (D) Induction of apoptosis in HTLV-1-infected T-cell lines by AG490. Cells were treated in the absence (open bar) or presence (solid bar) of 50 μM AG490 for 48 h and stained with Annexin-V. Apoptosis was analyzed by flow cytometry. Data represent mean percentages of apoptotic cells from three independent experiments.
Mentions: Next we examined the effect of AG490 on the growth of HTLV-1-infected T-cell lines and primary ATL cells. HTLV-1-infected T-cell lines were treated with different concentration of AG490 (0, 25 or 50 μM) and cell numbers were counted 24 and 48 h after treatment. AG490 suppressed the growth of HTLV-1-infected T-cell lines in a dose and time dependent manner (Figure 4A). The antiproliferative effects of AG490 against primary ATL cells and PBMCs from healthy donors were measured by WST-8 method (Cell Counting Kit-8; Wako Chemical, Osaka, Japan) based on the MTT assay as described previously [20]. Cell viability was determined as percentage of the control (without AG490). AG490 also inhibited the growth of PBMCs from ATL patients (ATL #1–7 in Figure 4B). In comparison, the cell growth inhibitory effect on PBMCs from healthy donors was weak (Normal #1–3 in Figure 4B). These findings indicate that AG490 inhibits the growth of cells infected with HTLV-1 but not that of uninfected PBMCs.

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