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To shed light on the molecular mechanisms associated with aberrant accumulation of c-Myb in chronic myeloid leukemia, comparative proteomic analysis was performed on c-myb RNAi-specifically silenced K562 cells, sampled on a time-course basis. 2D-DIGE technology highlighted 37 differentially-represented proteins that were further characterized by nLC-ESI-LIT-MS/MS and validated by western blotting and qRT-PCR analysis. Most of the deregulated proteins were related to protein folding, energy/primary metabolism, transcription/translation regulation and oxidative stress response. Protein network analysis suggested that glycolysis, gluconeogenesis and protein ubiquitination biosynthesis pathways were highly represented, confirming also the pivotal role of c-Myc. A specific reduced representation was observed for glyceraldehyde-3-phosphate-dehydrogenase and α-enolase, suggesting a possible role of c-Myb in the activation of aerobic glycolysis. A reduced amount was also observed for stress responsive heat shock 70kDa protein and 78kDa glucose-regulated protein, previously identified as direct targets of c-Myb. Among over-represented proteins, worth mentioning is the chromatin modifier chromobox protein homolog 3 that contributes to silencing of E2F- and Myc-responsive genes in quiescent G0 cells. Data here presented, while providing novel insights onto the molecular mechanisms underlying c-Myb activity, indicate potential protein biomarkers for monitoring the progression of chronic myeloid leukemia., Biological Significance: Myeloid leukemia is a malignant disease of the hematopoietic system in which cells of myeloid lineages accumulate to an undifferentiated state. In particular, it was shown that an aberrant accumulation of the c-Myb transcriptional factor is associated with the suppression of normal differentiation processes promoting the development of the hematopoietic malignancies. Many efforts have been recently made to identify novel genes directly targeted by c-Myb at a transcriptome level. In this work, we originally describe a differential proteomic approach to facilitate the comprehension of the regulation of the protein networks exerted by c-Myb. Our study reveals a complex network of proteins regulated by c-Myb. The functional heterogeneity of these proteins emphasizes the pleiotropic role of c-Myb as a regulator of genes that are crucial for energy production and stress response in leukemia. In fact, variations in glyceraldehyde-3-phosphate-dehydrogenase and α-enolase suggest a possible role of c-Myb in the activation of aerobic glycolysis. Moreover, significant differences were found for heat shock 70kDa protein and 78kDa glucose-regulated protein known as direct c-Myb targets. This work highlights potential protein biomarkers to look into disease progression and to develop translational medicine approaches in myeloid leukemia., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The p53 gene is rarely mutated in neuroblastoma, but codon 72 polymorphism that modulates its proapoptotic activity might influence cancer risk and clinical outcome. We investigated whether this polymorphism affects neuroblastoma risk and disease outcome and assessed the biologic effects of the p53-72R and p53-72P isoforms in p53-null cells. Comparison of 288 healthy subjects and 286 neuroblastoma patients revealed that the p53-72 polymorphism had no significant impact on the risk of developing neuroblastoma; however, patients with the Pro/Pro genotype had a shorter survival than those with the Arg/Arg or the Arg/Pro genotypes even in the stage 3 and 4 subgroup without MYCN amplification. By Cox regression analysis, the p53 Pro/Pro genotype seems to be an independent marker of poor prognosis (hazard ratio = 2.74; 95% confidence interval = 1.14-6.55, P = .014) together with clinical stage, MYCN status, and age at diagnosis. In vitro, p53-72P was less effective than p53-72R in inducing apoptosis and inhibiting survival of p53-null LAN-1 cells treated with etoposide, topotecan, or ionizing radiation but not taxol. By contrast, p53-72P was more effective in promoting p21-dependent accelerated senescence, alone or in the presence of etoposide. Thus, the p53-72 Pro/Pro genotype might be a marker of poor outcome independent of MYCN amplification, possibly improving risk stratification. Moreover, the lower apoptosis and the enhanced accelerated senescence by the p53-72P isoform in response to DNA damage suggest that patients with neuroblastoma with the p53-72 Pro/Pro genotype may benefit from therapeutic protocols that do not rely only on cytotoxic drugs that function, in part, through p53 activation.

We tested the activity of a p53 carboxy-terminal peptide containing the PARC-interacting region in cancer cells with wild type cytoplasmic p53. Peptide delivery was achieved by fusing it to the TAT transduction domain (TAT-p53-C-ter peptide). In a two-hybrid assay, the tetramerization domain (TD) of p53 was necessary and sufficient to bind PARC. The TAT-p53-C-ter peptide disrupted the PARC-p53 complex. Peptide treatment caused p53 nuclear relocation, p53-dependent changes in gene expression and enhancement of etoposide-induced apoptosis. These studies suggest that PARC-interacting peptides are promising candidates for the enhancement of p53-dependent apoptosis in tumors with wt cytoplasmic p53.

The insulin-like growth factor (IGF) axis is frequently activated in neuroblastoma (NB) tumors and cell lines. We show that silencing endogenous expression of IGF Binding Protein-5 (IGFBP-5) in NB cells by using microRNA and siRNA causes mitochondrial apoptosis that is characterized by: (a) release of cytochrome C in the cytoplasm and activation of caspase 9; (b) Erk1 and Erk2 inhibition; and (c) upregulation of pro-apoptotic proteins Bim and Bax. Bim upregulation is caused, at least in part, by protein stabilization that may depend on inhibition of Erk1 and Erk2. Of interest, Bim knock-down by siRNA decreases apoptosis in IGFBP-5-interfered cells. Thus, inhibition of endogenously produced IGFBP-5 is associated with Bim-dependent apoptosis in NB cells.