Figure 6. BMSCs secrete soluble factor(s) that decrease ENT1 nucleotide transport protein activity by removing ENT1 from the cell surface.

THP-1 cells (A) or AML patient samples (B) were cultured alone or with human BMSC SN from HS5 (SN HS5) for 24 hours. Cells were harvested and hENT1 activity was measured by the incorporation of radioactive ³H-adenosine. Each bar represents the mean ± SD of 3 independent experiments. ***p < 0.001 (THP1 versus THP1 in HS5 SN). *p < 0.05 (patient AML versus patient AML cultured HS5). (C) RNA extraction from THP1 cells cultured alone or in presence of human BMSC SN from HS5 (SN HS5) for 24 hours was used for RT-PCR to detect expression of ENT1. (D) The ENT1 protein from THP1 cells disappears from the cell surface by BMSC SN HS5 treatment. TPH1 cells were incubated with human BMSC SN from HS5 (BM SN HS5) for the indicated time periods at 37°C and were subjected to cell surface biotinylation at 4°C. The biotinylated proteins were then precipitated using Neutroavidin-Sepharose, resolved in SDS-PAGE and analyzed by immunoblot with anti-ENT1 antibodies. Total ENT1 corresponds to the immunoblot of 20% of the total cell extract. BM SN HS5 treatment induced removal of ENT1 from the cell surface in the first 2 hours and extending to the 24 hours of the experiment, without major changes in the ENT1 molecular mass.