A Role for Survivin in Enucleation of Erythroid Progenitors.

Abstract

Enucleation is the final step in the development of reticulocytes and this process is unique to mammalian red blood cells among vertebrates. Even though enucleation of erythroblasts was structurally studied by electron microscopy half a century ago, little is known about the underlying mechanism. Initially it was thought that enucleation involved degradation of DNA and other apoptotic processes. More recently it has been shown that enucleation of erythroid progenitors does not require major apoptotic machinery including caspase-3 activation (Krauss et al, 2005; Yoshida et al, 2005). Alternatively, it has been suggested that enucleation in erythroblasts is a result of asymmetric cytokinesis. In support of this later model, survivin, a regulator of chromosomal segregation and cytokinesis, which also has an anti apoptotic role, is highly expressed in orthochromatic erythroblasts, cells at the stage of differentiation just prior to enucleation (Gurbuxani et al, 2005). Moreover conditional heterozygous deletion of survivin in hematopoietic cells caused a decrease in enucleated cells in the spleen (Leung et al, 2007). This has led us to hypothesize that survivin has a role in enucleation of erythroblasts. To test this prediction, we first over expressed survivin in MEL (murine erythroleukemia) cells and then subjected them to DMSO induced differentiation. Normally MEL cells decrease their survivin expression level upon differentiation and at the same time undergo limited enucleation (<4%) even after 5 days of differentiation. In contrast, survivin overexpressing clones consistently enucleated 3–5 fold more than control cells, suggesting that levels of survivin are likely to be important for enucleation. We are currently harnessing the power of survivin antisense nanoparticle agents to assess the specific requirement of survivin in enucleation of primary human orthochromatic erythroblasts. Separately, we evaluated the localization of survivin and its known partners in enucleating primary human erythroid cells by confocal microscopy. Survivin is predominantly localized to the cytoplasmic compartment and to our surprise did not colocalize with Aurora B kinase and INCENP, two of its partners in the chromosomal passenger complex, which is essential for mitosis. On the contrary, we noticed that survivin partially colocalized with actin at the junction between the extruding nucleus and cytoplasm, as well as in the periphery of cytoplasm in these enucleating cells. Moreover enucleation assays performed in the presence of various inhibitors of cell cycle and cytokinesis did not prevent enucleation in erythroblasts. Taken together, these findings demonstrate that enucleation is not a form of cytokinesis and further suggest that survivin participates in enucleation by a novel mechanism.