Abstract
E2A, a basic helix loop helix transcription factor, is essential for B lymphocyte proliferation, survival and differentiation. Mutations in E2A are associated with B cell leukemia and lymphomas. Our preliminary data suggests that E2A activity is altered by HSP70 and since HSP70 ATPase activity is inhibited by methylene blue (MB), we hypothesized that MB will alter E2A activity and thus alter B cell development.
We studied the effects of MB on normal and leukemic B cells compared to its effect on myeloid cells. The cell lines used were REH, NALM-6 and BAF3-p185 cells compared to myeloid cell lines, CCRF and CMK for the proliferation assay. To study MB effect on normal B and myeloid progenitors, methylcellulose colony formation assays with murine bone marrow cells (strain 129S1/SVImJ, 8-12 weeks old mice) were performed. We also treated mice with MB in their drinking water (1000 μM) up to 16 weeks to study the in-vivo effect on B cells as compared to granulocyte, macrophages, T cells and NK cells in the peripheral blood by flow cytometry analysis. To understand the mechanisms by which MB alters B cell proliferation, we examined the effects of MB on cell cycle progression, apoptosis and E2A activity in B cells. For cell progression NALM-6 cells were treated with 4 μM MB for 0, 6, 12 and 24 hrs and assayed using BRDU staining by flow cytometry analysis. Further, apoptosis was measured using Annexin V staining by flow cytometry analysis in NALM-6 cells up to 48hrs post treatment with MB. Lastly, E2A activity was measured using an E2A-specific luciferase reporter assay in 293T cells.
MB decreased the proliferation of precursor B cell lines, REH, NALM-6 and BAF3-p185 cells compared to myeloid cell lines, CCRF and CMK. Similar effects of MB were also seen with normal B and myeloid progenitors as assayed by methylcellulose colony formation assays with murine bone marrow cells. Similarly, there is a significant decrease in peripheral B cells as compared to granulocytes, macrophages, T cells and NK cells when the mice were treated with 1000 μM of methylene blue in their drinking water as compared to those that remained untreated. MB caused apoptosis and decreased the S phase fraction of treated NALM-6 cells. In addition, we found that MB inhibited E2A activity using an E2A-specific luciferase reporter assay.
Our findings suggest that MB is a specific inhibitor of B cell proliferation, survival and development. This effect may be mediated by the inhibition of E2A activity by methylene blue.
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
