Exploring the anti-apoptotic role of HAX-1 versus BCL-XL in cytokine-dependent bone marrow-derived cells from mice

AbstractHS-1-associated protein X-1 (HAX-1) is a multi-functional protein that has been implicated in the regulation of apoptosis, cell motility and calcium homeostasis. In the present study, we set out to assess the postulated functional resemblance of HAX-1 to the BCL-2 family of anti-apoptotic proteins using non-transformed, cytokine-dependent murine bone marrow cells as a model system. BCL-XL, but not HAX-1 protected against cytokine withdrawal-induced apoptosis while HAX-1 and BCL-XL significantly reduced thapsigargin-triggered (calcium-dependent) apoptosis. The data argue in favor of cell type- and stimulus-specific roles of HAX-1 in regulation of cell survival

Inhibition of the Intrinsic but Not the Extrinsic Apoptosis Pathway Accelerates and Drives Myc-Driven Tumorigenesis Towards Acute Myeloid Leukemia

Myc plays an important role in tumor development, including acute myeloid leukemia (AML). However, MYC is also a powerful inducer of apoptosis, which is one of the major failsafe programs to prevent cancer development. To clarify the relative importance of the extrinsic (death receptor-mediated) versus the intrinsic (mitochondrial) pathway of apoptosis in MYC-driven AML, we coexpressed MYC together with anti-apoptotic proteins of relevance for AML; BCL-XL/BCL-2 (inhibiting the intrinsic pathway) or FLIPL (inhibiting the extrinsic pathway), in hematopoietic stems cells (HSCs). Transplantation of HSCs expressing MYC into syngeneic recipient mice resulted in development of AML and T-cell lymphomas within 7–9 weeks as expected. Importantly, coexpression of MYC together with BCL-XL/BCL-2 resulted in strongly accelerated kinetics and favored tumor development towards aggressive AML. In contrast, coexpression of MYC and FLIPL did neither accelerate tumorigenesis nor change the ratio of AML versus T-cell lymphoma. However, a change in distribution of immature CD4+CD8+ versus mature CD4+ T-cell lymphoma was observed in MYC/FLIPL mice, possibly as a result of increased survival of the CD4+ population, but this did not significantly affect the outcome of the disease. In conclusion, our findings provide direct evidence that BCL-XL and BCL-2 but not FLIPL acts in synergy with MYC to drive AML development

Blast formation of Mock-GFP/Mock-YFP, Mock-GFP/MYC-YFP or BCL-X_L-GFP/MYC-YFP bone marrow and spleen cells.

<p>(A) Forward light scatter of CD11b⁺Gr1⁺ bone marrow cells from Mock-GFP/Mock-YFP, Mock-GFP/MYC-YFP and BCL-X_L-GFP/MYC-YFP recipient mice 2 weeks after transplantation. Grey thick lines indicate GFP⁻YFP⁺ cells, thick black lines indicate GFP⁺YFP⁻ cells and thin lines indicate GFP⁺YFP⁺ cells. (B) Difference in mean fluorescence intensity (Δmfi) in forward scatter of CD11b⁺Gr1⁺ (left), CD19⁺IgM⁻ (middle) and CD19⁺IgM⁺ (right) cells in bone marrow (top) and spleen (bottom) between GFP⁻YFP⁻ (non-transduced cells) and GFP⁻YFP⁺ (black bars), GFP⁺YFP⁺ (striped bars) or GFP⁺YFP⁻ (white bars) cells are shown. Values indicate means of three individual mice and error bars indicate 1 SD.</p

Overexpression of MYC in HSC induces both myeloid and lymphoid leukemia.

<p>Flow cytometry analysis of bone marrow and spleen cells from two individual moribund Mock/MYC mice (indicated with # in the left). In the middle, percentage of GFP⁺YFP⁺ (Mock/MYC expressing) cells or GFP⁻YFP⁺ (MYC expressing) cells are indicated. These cells were further characterized with anti-CD11b, anti-Gr1, anti-CD4, anti-CD8, anti-CD19, anti-IgM anti-CD71 and anti-Ter119. The percentage of cells in each quadrant is indicated.</p

Overexpression of BCL-X_L-GFP/MYC-YFP induces AML.

<p>Flow cytometry analysis of bone marrow (top), spleen (middle) and liver (bottom) cells from one representative moribund BCL-X_L-GFP/MYC-YFP mouse. Single cell suspensions from femoral bone marrow, spleen and liver were stained with anti-CD11b and anti-Gr1. The percentage of dominating cells is indicated.</p

BCL-X_L and BCL-2 but not FLIP_L accelerate Myc-induced hematopoietic tumorigenesis.

<p>Kaplan-Meier survival analysis of mice transplanted with HSC over-expressing (A) Mock-GFP/MYC-YFP in DBA/2, (B) BCL-X_L -GFP/MYC-YFP in DBA/2, (C) FLIP_L-GFP/MYC-YFP in DBA/2, (D) BCL-X_L-GFP/MYC-YFP in BALB/c and (E) BCL-2-GFP/MYC-YFP in BALB/c. Mice were monitored daily for tumors or signs of paralysis and killed if showing signs of sickness. The percentage of mice surviving at daily intervals is shown.</p

Tumor phenotype in Mock/MYC, FLIP_L/MYC, BCL-X_L/MYC and BCL-2/MYC recipient mice.

<p>Summary of flow cytometry analysis of spleen cells from moribund mice transplanted with HSC expressing (A) Mock-GFP/MYC-YFP into DBA/2 mice, (B) FLIP_L-GFP/MYC-YFP into DBA/2 mice, (C) BCL-X_L-GFP/MYC-YFP into DBA2 mice, (D) BCL-X_L-GFP/MYC-YFP into BALB/c mice and (E) BCL-2-GFP/MYC-YFP into BALB/c mice. Left panel specify tumors expressing MYC-YFP only and right panel specify cells expressing MYC-YFP together with either Mock-GFP (A), FLIP_L-GFP (B), BCL-X_L-GFP (C and D) or BCL-2-GFP (E). Filled box indicate tumor phenotype. M indicates tumors of myeloid lineage, DP indicate CD4⁺CD8⁺ lymphoid tumors, 4 indicate CD4⁺ lymphoid tumors and 8 indicate CD8⁺ lymphoid tumors. Numbers correspond to identity of individual animal.</p

Statistical analysis of survival data after transplantation of hematopoietic stem cells expressing MYC, BCL-X_L, BCL-2 and/or FLIP_L.

<p>Statistical analysis of the survival data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031366#pone-0031366-g002" target="_blank">Figure 2</a>.</p>*<p>Log-rank (Mantel-Cox) test. NS = non significant. S = significant. P-value indicated below.</p