GTPase Activating Protein (Phospho-Ser387) ELISA Kit

The stem cell-intrinsic model of self-renewal via asymmetric cell division (ACD) posits that fate determinants be partitioned unequally between daughter cells to either activate or suppress the stemness state. ACD is a purported mechanism by which hematopoietic stem cells (HSCs) self-renew, but definitive evidence for this cellular process remains open to conjecture. To address this issue, we chose 73 candidate genes that function within the cell polarity network to identify potential determinants that may concomitantly alter HSC fate while also exhibiting asymmetric segregation at cell division. Initial gene-expression profiles of polarity candidates showed high and differential expression in both HSCs and leukemia stem cells. Altered HSC fate was assessed by our established in vitro to in vivo screen on a subcohort of candidate polarity genes, which revealed 6 novel positive regulators of HSC function: Ap2a2, Gpsm2, Tmod1, Kif3a, Racgap1, and Ccnb1. Interestingly, live-cell videomicroscopy of the endocytic protein AP2A2 shows instances of asymmetric segregation during HSC/progenitor cell cytokinesis. These results contribute further evidence that ACD is functional in HSC self-renewal, suggest a role for Ap2a2 in HSC activity, and provide a unique opportunity to prospectively analyze progeny from HSC asymmetric divisions. (Blood. 2012; 119(11):2510-2522)

There are two major pathways leading to induction of NF-kappa B subunits. The classical (or canonical) pathway typically leads to the induction of RelA or c-Rel containing complexes, and involves the degradation of I kappa B alpha in a manner dependent on I kappa B kinase (IKK) beta and the IKK regulatory subunit NEMO. The alternative (or non-canonical) pathway, involves the inducible processing of p100 to p52, leading to the induction of NF-kappa B2(p52)/RelB containing complexes, and is dependent on IKK alpha and NF-kappa B inducing kinase (NIK). Here we demonstrate that in primary human fibroblasts, the alternative NF-kappa B pathway subunits NF-kappa B2 and RelB have multiple, but distinct, effects on the expression of key regulators of the cell cycle, reactive oxygen species (ROS) generation and protein stability. Specifically, following siRNA knockdown, quantitative PCR, western blot analyses and chromatin immunoprecipitation (ChIP) show that NF-kappa B2 regulates the expression of CDK4 and CDK6, while RelB, through the regulation of genes such as PSMA5 and ANAPC1, regulates the stability of p21WAF1 and the tumour suppressor p53. These combine to regulate the activity of the retinoblastoma protein, Rb, leading to induction of polycomb protein EZH2 expression. Moreover, our ChIP analysis demonstrates that EZH2 is also a direct NF-kappa B target gene. Microarray analysis revealed that in fibroblasts, EZH2 antagonizes a subset of p53 target genes previously associated with the senescent cell phenotype, including DEK and RacGAP1. We show that this pathway provides the major route of crosstalk between the alternative NF-kappa B pathway and p53, a consequence of which is to suppress cell senescence. Importantly, we find that activation of NF-kappa B also induces EZH2 expression in CD40L stimulated cells from Chronic Lymphocytic Leukemia patients. We therefore propose that this pathway provides a mechanism through which microenvironment induced NF-kappa B can inhibit tumor suppressor function and promote tumorigenesis.