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p66shc deletion exacerbates leukemia in TCL1 mice
tively, compared to that of leukemic Eμ-TCL1 cells, as assessed in transwell assays (Online Supplementary Figure S7). Although surface and mRNA levels of CXCR4 were similar in the two mouse strains, tumoral Eμ- TCL1/p66Shc-/- cell chemotaxis towards the CXCR4 lig- and CXCL12 was enhanced (Online Supplementary Figure S7), consistent with the ability of p66Shc to negatively regulate CXCR4-dependent signaling9 and CXCR4 recy- cling7 in human B cells.
Lymphocyte homing to non-lymphoid organs is con- trolled by G protein-coupled receptors. B-cell homing to the liver is regulated by CCR1, CCR2, and CXCR3, while CCR2, CCR5, and CXCR3 have been implicated in B-cell homing to the lung.29-34 CCR1 and CCR5 mRNA levels in leukemic cells were comparable in the two mouse strains (Online Supplementary Figure S8). Conversely, both surface and mRNA levels of CCR2 and CXCR3 were higher in leukemic Eμ-TCL1/p66Shc-/- cells
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Figure 3. p66Shc deficiency in leukemic cells results in enhanced chemoresis- tance. (A) Quantitative real-time poly- merase chain reaction analysis of Bcl-2 and Bax mRNA in leukemic cells purified from either wildtype (WT) (n=7) or p66Shc-/- (n=7) mice and from Eμ-TCL1 (n=10) or Eμ-TCL1/p66Shc-/- (n=12) mice with overt leukemia. The relative gene transcript abundance was deter- mined on triplicate samples using the DDCt method. (B) Immunoblot analysis with anti-Bcl-2 (left) and anti-Bax (right) antibodies of postnuclear supernatants of leukemic cells purified from either WT (n=3) or p66Shc-/- (n=3) mice and from Eμ-TCL1 (n=3) or Eμ-TCL1/p66Shc-/- (n=3) mice with overt leukemia. The stripped filters were reprobed with anti- actin antibodies. (C) Flow cytometric analysis of the percentages of annexin V+CD5+IgM+ cells in peripheral blood from either WT (n=9) or p66Shc-/- (n=8) mice and from Eμ-TCL1 (n=20) or Eμ- TCL1/p66Shc-/- (n=22) mice. Samples were treated with either dimethylsulfox- ide (DMSO) or 35 μM fludarabine (flu) for 16 h at 37°C. (D) Flow cytometric analysis of the percentages of annexin V+CD5+IgM+ cells in peripheral blood from either Eμ-TCL1 (n=20) or Eμ- TCL1/p66Shc-/- (n=22) mice with <35% (black boxes) or ≥35% (gray boxes) CD5+CD19+ leukemic cells in peripheral blood, treated with either DMSO or 35 μM fludarabine for 16 h at 37°C. Mean ± standard deviation. One-way analysis of variance (ANOVA), multiple compar- isons. ****P≤0.0001; ***P≤0.001; **P≤0.01; *P≤0.05
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haematologica | 2019; 104(10)
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