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Bone marrow adherent layers inhibit apoptosis of acute myeloid leukemia cells
L J Bendall1, A Daniel, K Kortlepel
1Department of Haematology, Westmead Hospital, Sydney, Australia.
Abstract:
Human acute myeloid leukemia (AML) cells, like normal hematopoietic progenitors, die rapidly by apoptosis when cultured under serum-free conditions. Apoptosis was demonstrated by electron microscopy and agarose gel electrophoresis and quantified by flow cytometry. Culturing AML blasts in the presence of a bone marrow fibroblast (BMF) monolayer reduced the percentage of AML blasts undergoing apoptosis in the majority of cases studied. The effect was more pronounced when AML cells were cultured in the presence of an adherent long-term bone marrow (LTBM) stroma rather than BMF. Overall, the mean percentage of AML cells with fragmented DNA fell from 85 +/- 8% in control cultures to 20 +/- 9% in cultures with adherent stroma (p = 0.0004, n = 7). Supplementation of serum-free medium with recombinant cytokines, including stem cell factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF)-alpha or with human placenta-conditioned medium (HPCM) matched the degree of inhibition of apoptosis induced by BMF in only 50% of cases. Granulocyte colony-stimulating factor (G-CSF), interleukin-1 beta (IL-1 beta), and IL-6 were completely ineffective. Consistent with this observation, direct contact between leukemic cells and adherent layers was essential for maximum inhibition of leukemic-cell DNA fragmentation. Separation by a porous membrane allowing passage of soluble growth factors, but interrupting direct cell contact, was associated with significantly greater DNA fragmentation and cell death. Inhibition of leukemic-cell apoptosis correlated with improved survival and growth of malignant clonogenic cells. Colonies grown in cultures were identified as leukemic by morphology and by fluorescence in in situ hybridization to demonstrate numerical chromosomal abnormalities identified at diagnosis. Close contact between leukemic cells and bone marrow inhibits blast cell apoptosis and directly promotes survival of clonogenic AML cells.
Insights
Bone marrow stroma inhibits apoptosis in human acute myeloid leukemia (AML) cells. Direct cell contact is crucial for this effect, promoting survival of malignant clonogenic cells.
Area of Science:
- Hematology
- Cancer Biology
- Cell Biology
Background:
- Human acute myeloid leukemia (AML) cells undergo rapid apoptosis in serum-free conditions, similar to normal hematopoietic progenitors.
- The bone marrow microenvironment plays a critical role in regulating hematopoietic cell survival and proliferation.
Purpose of the Study:
- To investigate the role of bone marrow stromal cells and direct cell contact in regulating apoptosis of human AML cells.
- To determine the impact of stromal support on the survival of clonogenic AML cells.
Main Methods:
- AML cells were cultured under serum-free conditions with or without bone marrow fibroblast (BMF) monolayers or adherent long-term bone marrow (LTBM) stroma.
- Apoptosis was assessed by electron microscopy, agarose gel electrophoresis, and flow cytometry (DNA fragmentation).
- The effect of recombinant cytokines and conditioned medium on AML cell apoptosis was evaluated. Direct cell contact was manipulated using porous membranes.
Main Results:
- Adherent LTBM stroma significantly inhibited AML cell apoptosis, reducing DNA fragmentation from 85% to 20% (p = 0.0004).
- Direct contact between AML cells and stromal layers was essential for maximum inhibition of apoptosis.
- Recombinant cytokines partially mimicked the anti-apoptotic effect of BMF, but direct contact was superior.
- Inhibition of apoptosis correlated with improved survival and growth of malignant clonogenic AML cells.
Conclusions:
- Close contact with bone marrow stroma inhibits blast cell apoptosis in human AML.
- This direct interaction promotes the survival of clonogenic AML cells, suggesting a critical role for the bone marrow microenvironment in AML pathogenesis.
- Targeting cell-stroma interactions could represent a therapeutic strategy for AML.