APC/C-Cdh1 as a Novel Regulator of Hematopoietic Stem Cell Differentiation.

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that regulates cell cycle progression. This is achieved by targeting various cell cycle regulators for proteasomal destruction. APC/C in conjunction with its adaptor protein Cdh1, both stabilizes G1-Phase (a pre-condition for an accurate cell cycle progression) and is involved in the induction of cell cycle arrest and differentiation. Further evidence suggests that Cdh1 is involved in the differentiation of a variety of cells such as neurons, myocytes, hepatocytes and lens epithelial cells. During differentiation Cdh1 interacts with the TGFb signaling pathway, targets Id2 for destruction and indirectly leads to accumulation of p27 by Skp2 degradation. We have been able to demonstrate that the expression of Cdh1 is decreased in both, AML cell lines and in primary blast samples carrying the translocation t(8;21), which consequently leads to an AML1/Eto fusion protein and is one of the most common chromosomal rearrangements in AML. Furthermore, we have data suggesting that APC/C-Cdh1 significantly influences the differentiation of malignant myeloid cells. Here, we have analyzed the expression of Cdh1, its target proteins and relevant cell cycle regulators during normal myeloid differentiation.

A cytokine cocktail consisting of SCF (50ng/ml), IL-3 (5 ng/ml) and G-CSF (100 ng/ml) was used to induce differentiation of CD34+ cells into CD11b+ macrophage-like cells over seven days. Daily protein isolation, CD11b-FACS and FACS analysis of propidium iodide staining were performed to analyze Cdh1 status, differentiation kinetics and cell cycle distribution. In addition, we have established a Cdh1 knockdown in CD34+ cells by lentiviral vector mediated RNA interference. By means of GFP-cell-sorting the initially achieved transduction efficiency of 30% in CD34+ cells was increased to 70–80%.

The differentiation experiments carried out with normal CD34+ cells showed that after 7 days of stimulation the predominant majority of them had lost the CD34 marker and about 30% expressed CD11b on their surface confirming previous results. During the differentiation process an initial rise in Cdh1 levels, followed by a continuously high expression, was observed. Furthermore, we detected a downregulation of the Cdh1 target proteins Id2 and Skp2 and stable protein levels of p27. The cell cycle profile indicated an initial proliferation with an incremental G2/M-peak and at day 6 increasing apoptosis with a high Sub-G1-peak. In the transduced CD34+ cells we have been able to confirm by Western blotting analysis and RT-PCR that the CD34 positive cells harboring a Cdh1 shRNA had significantly decreased protein and RNA levels of Cdh1 compared to CD34 positive cells harboring a control shRNA against GFP. Analysis of the influence of Cdh1 knockdown on differentiation of CD34+ cells is ongoing and will be presented at the meeting.

These results are consistent with the important role of Cdh1 in initiating differentiation and also show its sustained function in post mitotic myeloid cells. Studying the differentiation characteristics of CD34+ cells with a Cdh1 knockdown is likely to help to further determine its function.

No relevant conflicts of interest to declare.