Abstract
We previously reported that TEL-FGFR3 in a patient with peripheral T-cell lymphoma and AML conferred IL-3 independency to Ba/F3 cells and activates MAPKs, p38, PI3K, Stat3 and Stat5 through its constitutive tyrosine kinase (TK) activity in TEL-FGFR3 transfected Ba/F3 cells (TF-V5). Both FGFR3 TK specific inhibitor SU5402 and PI3K inhibitor LY294002 inhibited cell growth of TF-V5 in a dose dependent manner (IC50=5μM, 10μM respectively). SU5402(25μM) resulted in G1 arrest (90%) and induced 80% of apoptosis to TF-V5 after 24hr. LY294002(50μM) decreased expression of c-Myc, Cyclin D3 and CDK4 in TF-V5 and induced G1 arrest (90%) and apoptosis about 20% to TF-V5 after 24hr. SU5402 completely suppressed expression of Bcl-xL and Bcl-2 after 24hr. LY294002 partially suppressed these expressions. As LY294002 did not affect the phosphorylation of STAT5 and STAT3, we hypothesized that these distinct apoptosis inducing abilities between SU5402 and LY294002 might be caused by the difference of Bcl-xL and Bcl-2 expression levels. Recent report suggests that FGFR3 activates Stat5 through recruitment of Pyk2 (proline-rich tyrosine kinase 2)-Src to its juxta-membrane (JM) domain. Therefore, we examined whether PP1/PP2 which are known as Src kinase inhibitors inhibit TF-V5 cell growth. PP1 and PP2 inhibited TEL-FGFR3 induced cell proliferation in a dose dependent manner (IC50=15μM, 15μM respectively). In contrast, growth of mock with IL-3 was not inhibited at a high concentration of PP1or PP2(30μM). PP1/PP2 did not affect auto-phosphorylation of TEL-FGFR3. Interestingly, PP1/PP2 inhibit Tyr phosphorylation of Pyk2 except Tyr402 and markedly suppressed activation of Stat3, Stat5 without affecting MAPKs and PLC gamma activation. PP1/PP2 suppressed expression of Bcl-xL and Bcl-2 partially but not of Bax and induced apoptosis 20% and 30% respectively. PP1/PP2 partially blocks cell cycle at G1 phase. When TF-V5 was co-treated with PP1(30μM)+LY294002(50μM) or PP2(30μM)+LY294002(50μM), apoptosis was induced in 60–70% of TF-V5 cells which remained at G1 phase after 24hr. Although PP1+LY294002 or PP2+LY294002 did not suppress Bcl-xL and Bcl-2 completely like SU5402, PP1/PP2 induced N-terminal cleavage form of Bax.
Conclusion: PP1/PP2 induces apoptosis in TF-V5 by activating Bax and by Pyk2-Src kinase inactivation that leads to the change of Bax/Bcl-2 or Bax/Bcl-XL ratio. Combination of PP1/PP2 and LY294002 induce high rate of apoptosis under G1 arrest in TF-V5 and may represent a novel approach against TEL-FGFR3 associated hematopoietic malignancies.
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