Georgios Nteliopoulos

Omega-3 polyunsaturated fatty acids selectively inhibit growth in neoplastic oral keratinocytes by differentially activating ERK1/2.

The long-chain omega-3 polyunsaturated fatty acids (n-3 PUFAs)—eicosapentaenoic acid (EPA) and its metabolite docosahexaenoic acid (DHA)—inhibit cancer formation in vivo, but their mechanism of action is unclear. Extracellular signal-regulated kinase 1/2 (ERK1/2) activation and inhibition have both been associated with the induction of tumour cell apoptosis by n-3 PUFAs. We show here that low doses of EPA, in particular, inhibited the growth of premalignant and malignant keratinocytes more than the growth of normal counterparts by a combination of cell cycle arrest and apoptosis. The growth inhibition of the oral squamous cell carcinoma (SCC) lines, but not normal keratinocytes, by both n-3 PUFAs was associated with epidermal growth factor receptor (EGFR) autophosphorylation, a sustained phosphorylation of ERK1/2 and its downstream target p90RSK but not with phosphorylation of the PI3 kinase target Akt. Inhibition of EGFR with either the EGFR kinase inhibitor AG1478 or an EGFR-blocking antibody inhibited ERK1/2 phosphorylation, and the blocking antibody partially antagonized growth inhibition by EPA but not by DHA. DHA generated more reactive oxygen species and activated more c-jun N-terminal kinase than EPA, potentially explaining its increased toxicity to normal keratinocytes. Our results show that, in part, EPA specifically inhibits SCC growth and development by creating a sustained signalling imbalance to amplify the EGFR/ERK/p90RSK pathway in neoplastic keratinocytes to a supraoptimal level, supporting the chemopreventive potential of EPA, whose toxicity to normal cells might be reduced further by blocking its metabolism to DHA. Furthermore, ERK1/2 phosphorylation may have potential as a biomarker of n-3 PUFA function in vivo.

Influence of PI-3K/Akt pathway on Wnt signalling in regulating myeloid progenitor cell proliferation. Evidence for a role of autocrine/paracrine Wnt regulation

The regulation of myeloid progenitor cell (granulocyte‐macrophage colony‐forming units, CFU‐GM) proliferation/differentiation by the Wnt and phosphatidylinositol‐3 kinase (PI‐3K) pathways was investigated using a colony‐replating assay. The PI‐3K pathway promoted differentiation of interleukin‐3 (IL‐3)‐stimulated myelopoiesis via Akt, because inhibition of the PI‐3K/Akt pathway with LY294002 or SH‐5 increased proliferation. The involvement of canonical and non‐canonical Wnt pathways was investigated using Wnt3a and Wnt5a respectively. Addition of the recombinant Wnts to IL‐3 increased CFU‐GM proliferation. Dkk‐1, when combined with the Wnt proteins, abrogated the effects of Wnt3a but not Wnt5a. Surprisingly, the addition of Dkk‐1 to LY294002 or SH‐5 blocked their proliferative effects. We hypothesized that increased proliferation induced by PI‐3K/Akt inhibitors was not mediated by downstream activation of the Wnt pathway but by induced endogenous production/release of Wnt proteins. The addition of SH‐5 to IL‐3 created an autocrine Wnt loop in CD34+ cells, resulting in the phosphorylation of lipoprotein‐receptor‐related‐protein 6. Furthermore, the addition of medium conditioned by CD34+ cells cultured in IL‐3 + SH‐5 to IL‐3 increased CFU‐GM proliferation. This effect was abrogated by Dkk‐1, suggesting that a Wnt in the conditioned medium increased proliferation. In summary, IL‐3 via the PI‐3K pathway promoted differentiation of myeloid progenitor cells through a decrease of endogenous Wnt production/release.

A Short-activating RNA Oligonucleotide Targeting the Islet β-cell Transcriptional Factor MafA in CD34+ Cells

Upon functional loss of insulin producing islet β-cells, some patients with diabetes become dependent on life-long insulin supplementation therapy. Bioengineering surrogate insulin producing cells is an alternative replacement strategy. We have developed a novel approach using short-activating RNA oligonucleotides to differentiate adult human CD34+ cells into insulin-secreting cells. By transfecting RNA to increase transcript levels of the master regulator of insulin biosynthesis, v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), several pancreatic endodermal genes were upregulated during the differentiation procedure. These included Pancreatic and duodenal homeobox gene-1 (PDX1), Neurogenin 3, NeuroD, and NK6 homeobox 1 (NKx6-1). Differentiated CD34+ cells also expressed glucokinase, glucagon-like peptide 1 receptor (GLP1R), sulfonylurea receptor-1 (SUR1) and phogrin—all essential for glucose sensitivity and insulin secretion. The differentiated cells appropriately processed C-peptide and insulin in response to increasing glucose stimulation as shown by enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting analysis, western blotting, and immunofluorescence staining. We provide a new approach using short-activating RNA in developing insulin producing surrogate cells for treating diabetes.

Antibody arrays identify protein-protein interactions in chronic myeloid leukaemia

Multiprotein complex formation with p210BCR‐ABL1 is likely to play a major role in determining cellular abnormalities in chronic myeloid leukaemia (CML). Although many p210BCR‐ABL1 binding partners have been identified, it is likely that many have not. We evaluated the use of co‐immunoprecipitation and antibody arrays and found that this approach is capable of identifying new p210BCR‐ABL1 binding partners, and may contribute to the search for new therapeutic targets in CML.

Exploiting Human CD34+ Stem Cell–conditioned Medium for Tissue Repair

Despite the progress in our understanding of genes essential for stem cell regulation and development, little is known about the factors secreted by stem cells and their effect on tissue regeneration. In particular, the factors secreted by human CD34+ cells remain to be elucidated. We have approached this challenge by performing a cytokine/growth factor microarray analysis of secreted soluble factors in medium conditioned by adherent human CD34+ cells. Thirty-two abundantly secreted factors have been identified, all of which are associated with cell proliferation, survival, tissue repair, and wound healing. The cultured CD34+ cells expressed known stem cell genes such as Nanog, Oct4, Sox2, c-kit, and HoxB4. The conditioned medium containing the secreted factors prevented cell death in liver cells exposed to liver toxin in vitro via inhibition of the caspase-3 signaling pathway. More importantly, in vivo studies using animal models of liver damage demonstrated that injection of the conditioned medium could repair damaged liver tissue (significant reduction in the necroinflammatory activity), as well as enable the animals to survive. Thus, we demonstrate that medium conditioned by human CD34+ cells has the potential for therapeutic repair of damaged tissue in vivo.

Protein Segregation Between Dividing Hematopoietic Progenitor Cells in the Determination of the Symmetry/Asymmetry of Cell Division

In the present study, we investigated how the symmetry/asymmetry of cell division in mitotic CD34(+) cells can be evaluated by determining the plane of cell division and the potential distribution of proteins between daughter cells. The orientation of the mitotic spindle is dependent upon the positioning of the centrosomes, which determine the plane of cell division and the sharing of proteins. If the functions of unequally shared proteins are relevant to the kinetics of cell division, they could determine whether the daughter cells undergo self-renewal or differentiation. The kinetic function of the proteins of interest was investigated using a colony-replating assay and carboxyfluorescein succinimidyl ester (CFSE) staining. We used Notch/Numb as a model system, since they have a role in balancing symmetric/asymmetric divisions. Mitotic cells were examined microscopically and centrosomal markers γ-tubulin/pericentrin were used with activated Notch-1 and Numb. We monitored the first crucial divisions by CFSE staining and found an inverse relationship between activated Notch and Numb expression, suggesting a reciprocal regulation. We suggest that the subpopulations expressing activated Notch or Numb have different cell fates. To determine the influence of Notch signaling on progenitor cell self-renewal, we used the γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-Butyl ester (DAPT). DAPT influences self-renewal/differentiation outcome by affecting the frequency of symmetric renewal divisions without affecting the rate of divisions. Overall, the purpose of this study was to establish a cellular system for predicting the symmetry/asymmetry of hematopoietic progenitor divisions at the level of centrosomes and protein distribution and to investigate the influence of these proteins on progenitor cell kinetics.