Direct activation of forkhead box O3 by tumor suppressors p53 and p73 is disrupted during liver regeneration in mice.

UNLABELLED: The p53 family of proteins regulates the expression of target genes that promote cell cycle arrest and apoptosis, which may be linked to cellular growth control as well as tumor suppression. Within the p53 family, p53 and the transactivating p73 isoform (TA-p73) have hepatic-specific functions in development and tumor suppression. Here, we determined TA-p73 interactions with chromatin in the adult mouse liver and found forkhead box O3 (Foxo3) to be one of 158 gene targets. Global profiling of hepatic gene expression in the regenerating liver versus the quiescent liver revealed specific, functional categories of genes regulated over the time of regeneration. Foxo3 is the most responsive gene among transcription factors with altered expression during regenerative cellular proliferation. p53 and TA-p73 bind a Foxo3 p53 response element (p53RE) and maintain active expression in the quiescent liver. During regeneration of the liver, the binding of p53 and TA-p73, the recruitment of acetyltransferase p300, and the active chromatin structure of Foxo3 are disrupted along with a loss of Foxo3 expression. In agreement with the loss of Foxo3 transcriptional activation, a decrease in histone activation marks (dimethylated histone H3 at lysine 4, acetylated histone H3 at lysine 14, and acetylated H4) at the Foxo3 p53RE was detected after partial hepatectomy in mice. These parameters of Foxo3 regulation are reestablished with the completion of liver growth and regeneration and support a temporary suspension of p53 and TA-p73 regulatory functions in normal cells during tissue regeneration. p53-dependent and TA-p73-dependent activation of Foxo3 was also observed in mouse embryonic fibroblasts and in mouse hepatoma cells overexpressing p53, TA-p73alpha, and TA-p73beta isoforms. CONCLUSION: p53 and p73 directly bind and activate the expression of the Foxo3 gene in the adult mouse liver and murine cell lines. p53, TA-p73, and p300 binding and Foxo3 expression decrease during liver regeneration, and this suggests a critical growth control mechanism mediated by these transcription factors in vivo.

ELMO1 and Dock180, a bipartite Rac1 guanine nucleotide exchange factor, promote human glioma cell invasion.

A distinct feature of malignant gliomas is the intrinsic ability of single tumor cells to disperse throughout the brain, contributing to the failure of existing therapies to alter the progression and recurrence of these deadly brain tumors. Regrettably, the mechanisms underlying the inherent invasiveness of glioma cells are poorly understood. Here, we report for the first time that engulfment and cell motility 1 (ELMO1) and dedicator of cytokinesis 1 (Dock180), a bipartite Rac1 guanine nucleotide exchange factor (GEF), are evidently linked to the invasive phenotype of glioma cells. Immunohistochemical analysis of primary human glioma specimens showed high expression levels of ELMO1 and Dock180 in actively invading tumor cells in the invasive areas, but not in the central regions of these tumors. Elevated expression of ELMO1 and Dock180 was also found in various human glioma cell lines compared with normal human astrocytes. Inhibition of endogenous ELMO1 and Dock180 expression significantly impeded glioma cell invasion in vitro and in brain tissue slices with a concomitant reduction in Rac1 activation. Conversely, exogenous expression of ELMO1 and Dock180 in glioma cells with low level endogenous expression increased their migratory and invasive capacity in vitro and in brain tissue. These data suggest that the bipartite GEF, ELMO1 and Dock180, play an important role in promoting cancer cell invasion and could be potential therapeutic targets for the treatment of diffuse malignant gliomas.

Generation and performance of an equine-specific large-scale gene expression microarray.

OBJECTIVE: To create high-quality sequence data for the generation of an equine gene expression microarray and evaluate array performance by use of lipopolysaccharide (LPS) exposure of synoviocytes. SAMPLE POPULATION: Public nucleotide sequence database from Equus caballus and synoviocytes from clinically normal adult horses. PROCEDURE: Computer procurement of equine gene sequences, probe design, and manufacture of an oligomicroarray were performed. Array performance was evaluated by use of patterns for equine synoviocytes in response to LPS. RESULTS: Starting with 18,924 equine gene sequences, 3,098 equine 3' sequences were annotated and met the inclusion criteria for an expression microarray. An equine oligonucleotide expression microarray was created by use of 68,266 of the 25-oligomer probes to uniquely identify each gene. Most genes in the array (68%) were expressed in equine synoviocytes. Repeatability of the array was high (r, > 0.99), and LPS upregulated (> 5-fold change) 84 genes, many of which were inflammatory mediators, and downregulated (> 5-fold change) 14 genes. An initial pattern of gene expression for effects of LPS on synoviocytes consisted of 102 genes. CONCLUSIONS AND CLINICAL RELEVANCE: Use of a computer algorithm to curate an equine sequence database generated high-quality annotated species-specific gene sequences and probe sets for a gene expression oligomicroarray, which was used to document changes in gene expression associated with LPS exposure of equine synoviocytes. The equine public database was expanded from 290 annotated genes to > 3,000 provisionally annotated genes. Similar curation and annotation of public databases could be used to create other species-specific microarrays.

Platelet-derived growth factor-B enhances glioma angiogenesis by stimulating vascular endothelial growth factor expression in tumor endothelia and by promoting pericyte recruitment.

Platelet-derived growth factor (PDGF)-B and its receptor (PDGF-R) beta are overexpressed in human gliomas and responsible for recruiting peri-endothelial cells to vessels. To establish the role of PDGF-B in glioma angiogenesis, we overexpressed PDGF-B in U87MG glioma cells. Although PDGF-B stimulated tyrosine phosphorylation of PDGF-Rbeta in U87MG cells, treatment with recombinant PDGF-B or overexpression of PDGF-B in U87MG cells had no effect on their proliferation. However, an increase of secreted PDGF-B in conditioned media of U87MG/PDGF-B cells promoted migration of endothelial cells expressing PDGF-R beta, whereas conditioned media from U87MG cells did not increase the cell migration. In mice, overexpression of PDGF-B in U87MG cells enhanced intracranial glioma formation by stimulating vascular endothelial growth factor (VEGF) expression in neovessels and by attracting vessel-associated pericytes. When PDGF-B and VEGF were overexpressed simultaneously by U87MG tumors, there was a marked increase of capillary-associated pericytes as seen in U87MG/VEGF(165)/PDGF-B gliomas. As a result of pericyte recruitment, vessels induced by VEGF in tumor vicinity migrated into the central regions of these tumors. These data suggest that PDGF-B is a paracrine factor in U87MG gliomas, and that PDGF-B enhances glioma angiogenesis, at least in part, by stimulating VEGF expression in tumor endothelia and by recruiting pericytes to neovessels.