MACS Technique Research Articles

Differences in the Localization of Receptor Tyrosine Kinase FLT3 in Human Hematopoietic Stem and Progenitor Cells and Leukemic Blasts.

Activation of type III receptor tyrosine kinase FLT3 by its ligand controls cell proliferation and differentiation in normal hematopoiesis. Constitutively activating mutations of FLT3 are common abnormalities in acute myeloid leukemia. Internal tandem duplication (ITD) mutations of the FLT3 receptor are found in approximately one third of patients with AML and normal karyotype and have been shown to be associated with inferior response towards chemotherapy. One possible way how mutant FLT3 receptors may be mediating its pathological effects is by altering the normal localization of the receptor protein. To determine the intracellular localization of wild type and mutant FLT3 receptors, confocal laser scanning microscopy was performed in cultured leukemic cell lines (THP-1, EOL-1, MV4-11) and primary hematopoietic stem cells (HSC) from healthy donors. HSC (CD34+) cells were prepared from human bone marrow and leukapheresis products of healthy donors by MACS technique using CD34 and CD135 (FLT3) coupled microbeads. Additionally, different leukemic cell lines were transduced with retroviral vectors encoding FLT3wt or FLT3-ITD receptors.In normal HSC, only 4–13% are expressing FLT3, and unexpectedly FLT3 staining was observed both in the plasma membrane and evenly distributed in the cytoplasm. The intracellular FLT3wt was not localized to the endoplasmic reticulum (ER), but part of it co-localized with the Trans-Golgi-network (TGN) and early endosomes.In leukemic cell lines endogenously expressing either wt or mutant FLT3, wtFLT3 localized to the plasma membrane and uniformly to the cytoplasm whereas mutant FLT3 strongly accumulated in a perinuclear region but did not localize to the plasma membrane. Stable transfection of leukemic cell lines with wt or mutant FLT3 confirmed this localization pattern. In leukemic cell lines neither FLT3wt nor FLT3-ITD accumulated in the ER or in the Golgi apparatus, but instead FLT3wt and FLT3-ITD seem to reside in the TGN and in early endosomes as shown by immunofluorescence costaining with the organelle markers.These data indicate that although perinuclear accumulation of mutant FLT3 is observed in all cells analyzed, wtFLT3 is also found intracellularly. FLT3-ITD might reside in a different state concerning glycosylation and/or phosphorylation (Schmidt-Arras et al., Mol Cell Biol, 2005) and therefore it may activate alternative or additional signaling cascades by having access to different or additional substrates. Further studies will clarify, whether intracellularly located wtFLT3 in healthy HSC and FLT3wt-expressing leukemic cells is also involved in FLT3 signaling.