The role of survivin in angiogenesis during zebrafish embryonic development

<p>Abstract</p> <p>Background</p> <p>Survivin is the smallest member of the inhibitor of apoptosis (IAP) gene family. Recently, the zebrafish <it>survivin-1 </it>gene has been cloned, showing remarkable sequence identity and similarity over the BIR domain compared with human and mouse <it>survivin </it>gene. Here we investigated the role of survivin in angiogenesis during zebrafish development. Morpholinos (MOs) targeting the 5' untranslated region (UTR) (Sur_UTR) and sequences flanking the initiation codon (Sur_ATG) of zebrafish <it>survivin-1 </it>gene were injected into embryos at 1–4 cell stage. Vasculature was examined by microangiography and GFP expression in <it>Tg(fli1:EGFP)</it>^<it>y</it>1embryos. Results: In embryos co-injected with Sur_UTRand Sur_ATG-MOs, vasculogenesis was intact but angiogenesis was markedly perturbed, especially in the inter-segmental vessels (ISV) and dorsal longitudinal anastomotic vessels (DLAV) of the trunk, the inner optic circle and optic veins of developing eyes and the sub-intestinal vessels. Apoptosis was increased, as shown by TUNEL staining and increase in caspase-3 activity. Efficacy of Sur_UTRand Sur_ATG-MOs was demonstrated by translation inhibition of co-injected 5'UTR survivin:GFP plasmids. The phenotypes could be recapitulated by splice-site MO targeting the exon2-intron junction of <it>survivin </it>gene and rescued by <it>survivin </it>mRNA. Injection of human vascular endothelial growth factor (VEGF) protein induced ectopic angiogenesis and increased survivin expression, whereas treatment with a VEGF receptor inhibitor markedly reduced angiogenesis and suppressed survivin expression. Conclusion: Survivin is involved in angiogenesis during zebrafish development and may be under VEGF regulation.</p

Overcoming myelosuppression due to synthetic lethal toxicity for FLT3-targeted acute myeloid leukemia therapy.

Activating mutations in FLT3 confer poor prognosis for individuals with acute myeloid leukemia (AML). Clinically active investigational FLT3 inhibitors can achieve complete remissions but their utility has been hampered by acquired resistance and myelosuppression attributed to a 'synthetic lethal toxicity' arising from simultaneous inhibition of FLT3 and KIT. We report a novel chemical strategy for selective FLT3 inhibition while avoiding KIT inhibition with the staurosporine analog, Star 27. Star 27 maintains potency against FLT3 in proliferation assays of FLT3-transformed cells compared with KIT-transformed cells, shows no toxicity towards normal human hematopoiesis at concentrations that inhibit primary FLT3-mutant AML blast growth, and is active against mutations that confer resistance to clinical inhibitors. As a more complete understanding of kinase networks emerges, it may be possible to define anti-targets such as KIT in the case of AML to allow improved kinase inhibitor design of clinical agents with enhanced efficacy and reduced toxicity

Overcoming myelosuppression due to synthetic lethal toxicity for FLT3-targeted acute myeloid leukemia therapy

Activating mutations in FLT3 confer poor prognosis for individuals with acute myeloid leukemia (AML). Clinically active investigational FLT3 inhibitors can achieve complete remissions but their utility has been hampered by acquired resistance and myelosuppression attributed to a 'synthetic lethal toxicity' arising from simultaneous inhibition of FLT3 and KIT. We report a novel chemical strategy for selective FLT3 inhibition while avoiding KIT inhibition with the staurosporine analog, Star 27. Star 27 maintains potency against FLT3 in proliferation assays of FLT3-transformed cells compared with KIT-transformed cells, shows no toxicity towards normal human hematopoiesis at concentrations that inhibit primary FLT3-mutant AML blast growth, and is active against mutations that confer resistance to clinical inhibitors. As a more complete understanding of kinase networks emerges, it may be possible to define anti-targets such as KIT in the case of AML to allow improved kinase inhibitor design of clinical agents with enhanced efficacy and reduced toxicity.published_or_final_versio

Sox4you:A New Player in C/EBPα Leukemia

Although CEBPA mutations are among the most common genetic abnormalities in acute myeloid leukemia (AML), the transformation mechanism remains largely obscure. In this issue of Cancer Cell, Zhang and colleagues report that SOX4 is a direct target and crucial mediator of C/EBPα mutants in AML, revealing a potential therapeutic avenue

All-trans retinoic acid induces proliferation of an irradiated stem cell supporting stromal cell line AFT024

OBJECTIVE: We have previously shown that all-trans retinoic acid (ATRA) enhanced the maintenance of early human hematopoietic progenitor cells (HPCs) in the presence of an irradiated stromal cell line AFT024. In this study, we examined the effects of ATRA on the stromal cell component with particular reference to cellular proliferation and gene expression. METHODS: Irradiated AFT024 cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with fetal bovine serum and were incubated with ATRA at 1 mumol/L up to 21 days. The cells were examined in terms of immunostaining for proliferative cell nuclear antigen (PCNA) and BrdU incorporation, apoptosis assay, cell cycle analysis, and gene expression using semiquantitative reverse-transcriptase polymerase chain reaction. RESULTS: In the control experiments, AFT024 cells lost their confluence in culture after 15-Gy irradiation and were arrested in G2/M phase on days 7 and 21. ATRA restored the cellular confluence with an increase in proliferation on day 21 (BrdU incorporation: 20.6-fold; PCNA staining: 51.7-fold) with reversal of cell cycle arrest (S phase: 2.7-fold increase; G2/M phase: 2.0-fold decrease). There was no effect on apoptosis as shown by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. ATRA significantly upregulated the expression of cell cycle genes for checkpoint transition, including cyclin A2, B2, and aurora kinase B, as well as genes associated with a putative role in HPC maintenance, including osteopontin, HoxA5, enhancer of zeste homolog 2, and peroxisome proliferator-activated receptor gamma. CONCLUSION: We concluded that ATRA induced cellular proliferation of irradiated AFT024 cells and expression of a number of genes whose relevance to HPC homeostasis would have to be further examined.status: publishe

Regulation of primitive hematopoiesis in zebrafish embryos by the death receptor gene

OBJECTIVE: We investigated the regulatory mechanism of primitive hematopoiesis in zebrafish (Danio rerio) embryos with particular reference to the role of a death receptor (zDR) gene, based on a morpholino (MO) knockdown approach. METHODS: MOs targeting the zDR and chordin (Chd) were injected into naturally spawned embryos at one- to four-cell stage. A random sequence (RS) MO was used as a control. Effects on hemoglobin formation (Hb), apoptosis, and lineage-specific gene expression were examined. Embryos injected with zDR, Chd, and RS-MOs were denoted zDR(mo), zChd(mo), and zRS(mo), respectively. Those co-injected with Chd+zDR-MOs and Chd+RS-MOs were abbreviated zChd+DR(mo) and zChd+RS(mo). RESULTS: zDR mRNA expression was restricted to the intermediate cell mass of wild-type (WT) and zChd(mo) embryos. At 48 hours postfertilization, zDR(mo) embryos showed increased Hb compared with WT or zRS(mo) embryos (2.36 x 10(-2) +/- 1.13 x 10(-3) vs 1.85 x 10(-2) +/- 5.60 x 10(-4) vs 1.79 x 10(-2) +/- 1.31 x 10(-3) U, p < 0.05). zChd+DR(mo) embryos also showed increased Hb compared with zChd(mo) or zChd+RS(mo) embryos (4.60 x 10(-2) +/- 2.79 x 10(-3) vs 3.17 x 10(-2) +/- 1.07 x 10(-3) vs 3.05 x 10(-2) +/- 1.25 x 10(-3) U, p < 0.05). zDR-MO reduced apoptosis, as shown by reduced terminal transferase-mediated dUTP nick end-labeling staining in zChd+DR(mo) compared with zChd+RS(mo) embryos and caspase-3 activity in zDR(mo) vs zRS(mo) (0.525 +/- 0.094 vs 0.953 +/- 0.113 U, p < 0.05), and zChd+DR(mo) vs zChd+RS(mo) embryos (0.247 +/- 0.121 vs 1.180 +/- 0.082, p < 0.05). zChd+DR(mo) embryos showed upregulation of erythroid-specific embryonic hemoglobin gene expression but not that of a myeloid-specific myeloperoxidase gene. CONCLUSION: Knockdown of zDR in zebrafish embryos decreased apoptosis and increased Hb, suggesting that zDR may regulate primitive hematopoiesis during development.status: publishe