We show here that a zinc finger transcriptional repressor, Slug, which is aberrantly upregulated by the E2A-HLF oncoprotein in pro-B cell acute leukemia, functions as an antiapoptotic factor in normal hematopoietic progenitor cells. Slug(-/-) mice were much more radiosensitive than wild-type mice, dying earlier and showing accentuated decreases in peripheral blood cell counts, as well as abundant microhemorrhages and widely disseminated bacterial microabscesses throughout the body. Slug expression was detected in diverse subsets of hematopoietic progenitors, but not in more differentiated B and T lymphoid cells, and there was a significant increase in apoptotic (TUNEL-positive) bone marrow progenitor cells in irradiated Slug(-/-) mice compared to wild-type controls. These results implicate Slug in a novel survival pathway that protects hematopoietic progenitors from apoptosis after DNA damage.

Increased levels of red cell fetal hemogloblin, whether due to hereditary persistence of expression or from induction with hydroxyurea therapy, effectively ameliorate sickle cell disease. Therefore, we developed erythroid-specific, γ-globin lentiviral vectors for hematopoietic stem cell-targeted gene therapy with the goal of permanently increasing HbF production in sickle red cells. We evaluated two different γ-globin lentiviral vectors for therapeutic efficacy in the BERK sickle cell mouse model. The first vector, V5, contained the γ-globin gene driven by 3.1 kb of β-globin regulatory sequences and a 130 bp β-globin promoter. The second vector, V5m3, was identical except that the γ-globin 3'UTR was replaced with the β-globin 3'UTR. Adult erythroid cells have β-globin mRNA 3’UTR binding proteins that enhance β-globin mRNA stability and we postulated this design might enhance γ-globin expression. Stem cell gene transfer was efficient and nearly all red cells in transplanted mice expressed human γ-globin. Both vectors demonstrated efficacy in disease correction, with the V5m3 vector producing a higher level of γ-globin mRNA which was associated with high level correction of anemia and secondary organ pathology. These data support the rationale for a gene therapy approach to sickle cell disease by permanently enhancing HbF using a γ-globin lentiviral vector.

Binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates a signaling cascade that causes ␣IIb␤3 activation and platelet aggregation. Previous work demonstrated that botrocetin (bt)/VWFmediated agglutination activates ␣IIb␤3 and elicits adenosine triphosphate (ATP) secretion in a thromboxane A2 (TxA2)-and Ca 2؉ -dependent manner. This agglutination-elicited TxA2 production occurs in the absence of ATP secretion. However, the signaling components and signaling network or pathway activated by GPIb-mediated agglutination to cause TxA2 production have not been identified. Therefore, the focus of this study was to elucidate at least part of the signal transduction network or pathway activated by GPIb-mediated agglutination to cause TxA2 production. The phosphatidylinositol 3-kinase (PI3K) selective inhibitor wortmannin, and mouse platelets deficient in Lyn, Src, Syk, Src homology 2 (SH2) domain-containing leukocyte protein 76 (SLP-76), phospholipase C␥2 (PLC␥2), linker for activation of T cells (LAT), or Fc receptor ␥-chain (FcR␥-chain) were used for these studies. LAT and FcR␥-chain were found not to be required for agglutination-driven TxA2 production or activation of ␣IIb␤3, but were required for granule secretion and aggregation. The results also clearly demonstrate that bt/VWF-mediated agglutination-induced TxA2 production is dependent on signaling apparently initiated by Lyn, enhanced by Src, and propagated through Syk, SLP-76, PI3K, PLC␥2, and protein kinase C (PKC) . IntroductionBinding of von Willebrand factor (VWF) to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates signaling that results in ␣IIb␤3 activation and platelet aggregation. [1][2][3][4] Interestingly, different modes of stimulation of the GPIb complex activate ␣IIb␤3 by apparently different mechanisms. For example, activation of ␣IIb␤3 in response to adhesion-independent shear stressinduced GPIb signaling (as in a cone and plate viscometer) requires Ca 2ϩ influx and probably mobilization of internal Ca 2ϩ stores, as well as adenosine diphosphate (ADP) secretion, but not thromboxane A2 (TxA2) production. [3][4][5][6] In contrast, activation of ␣IIb␤3 in response to adhesion-dependent shear stress-induced GPIb signaling (flow) does not require Ca 2ϩ influx (although Ca 2ϩ influx potentiates the process), 7 but does require mobilization of internal Ca 2ϩ stores, 2,7 and is not dependent on either ADP secretion or TxA2 production. 2,7 Likewise, adhesion-dependent, shear stressindependent GPIb-induced activation of ␣IIb␤3 appears to require mobilization of internal stores, but not Ca 2ϩ influx, ADP, or TxA2. 7,8 In further contrast to these systems, ␣IIb␤3 activation in response to botrocetin (bt)-facilitated, GPIb/VWF-mediated agglutination is dependent on TxA2 and the agglutination-elicited TxA2 production is not dependent on Ca 2ϩ influx or mobilization of internal Ca 2ϩ stores. 9 Despite the central role of agglutinationelicited TxA2 production in bt/VWF/GPIb-induced platelet acti...