Compatible solutes from hyperthermophiles improve the quality of DNA microarrays.

BACKGROUND: DNA microarrays are among the most widely used technical platforms for DNA and RNA studies, and issues related to microarrays sensitivity and specificity are therefore of general importance in life sciences. Compatible solutes are derived from hyperthermophilic microorganisms and allow such microorganisms to survive in environmental and stressful conditions. Compatible solutes show stabilization effects towards biological macromolecules, including DNA. RESULTS: We report here that compatible solutes from hyperthermophiles increased the performance of the hybridization buffer for Affymetrix GeneChip(R) arrays. The experimental setup included independent hybridizations with constant RNA over a wide range of compatible solute concentrations. The dependence of array quality and compatible solute was assessed using specialized statistical tools provided by both the proprietary Affymetrix quality control system and the open source Bioconductor suite. CONCLUSION: Low concentration (10 to 25 mM) of hydroxyectoine, potassium mannosylglycerate and potassium diglycerol phosphate in hybridization buffer positively affected hybridization parameters and enhanced microarrays outcome. This finding harbours a strong potential for the improvement of DNA microarray experiments.

GOAL: automated Gene Ontology analysis of expression profiles.

One of the most common problems encountered while deciphering results from expression profiling experiments is in relating differential expression of genes to molecular functions and cellular processes. A second important problem is that of comparing experiments performed by different labs using different microarray platforms, or even unrelated techniques. Gene Ontology (GO) is now used to describe biological features, since GO terms are associated with genes, to overcome the apparent distance between expression profiles and biological comprehension. Here we describe the development, implementation and use of GOAL (Gene Ontology Automated Lexicon), a web-based application for the identification of functions and processes regulated in microarray and SAGE (serial analysis of gene expression) experiments. We applied GOAL to a range of experimental datasets related to different biological problems, including cancer and the cell cycle. By using GOAL, reported and novel relevant processes were identified in a number of experiments by our collaborators and by us. Different datasets could also be compared with each other to define conserved functional modules. GOAL allows a seamless and high-level analysis of expression profiles and is implemented as a free WWW resource (http://microarrays.unife.it).

Identification of differentially expressed genes in human salivary gland tumors by DNA microarrays.

Differentially expressed genes among different benign and malignant salivary gland tumors were identified by use of cDNA microarrays containing 19,000 human expressed sequence tags. Tumors were classified by using a subset of 486 genes. Benign Warthin's tumor and pleomorphic adenoma showed very distinctive gene expression patterns. One hundred and thirty-three genes differentiated the single malignant clear cell carcinoma from non-tumor salivary glands (P < 0.01), whereas only 16 genes separated it from the highly related benign pleomorphic adenoma (P < 0.01). Fifty-seven cDNAs were associated with mucoepidermoid carcinoma (P < 0.01). The identified genes might help to disclose the molecular mechanisms and processes underlying malignant salivary gland tumors.

Molecular classification of nodal metastasis in primary larynx squamous cell carcinoma.

Classification and prognosis of larynx squamous cell carcinoma (LSCC) depends on clinical and histopathological examination. Currently, expression profiling harbors the potential to investigate, classify, and better manage cancer. Gene expression profiles of 22 primary LSCCs were analyzed by microarrays containing 19,200 cDNAs. GOAL functionally classified differentially expressed genes, and a novel "in silico" procedure identified physical gene clusters differentially transcribed. A signature of 158 genes differentiated tumors with nodal metastasis. A novel statistical method allowed categorization of metastatic tumors into 2 distinct subgroups of differential gene expression patterns. Among genes correlated to nodal metastatic progression, we verified in vitro that NM23-H3 reduced cell motility and TRIM8 were a growth suppressor. Six chromosomal regions were specifically downregulated in metastatic tumors. This large-scale gene expression analysis in LSCC provides information on changes in genomic activity associated with lymphonodal metastasis and identifies molecules that might prove useful as novel therapeutic targets.

Akt, protein kinase C, and mitogen-activated protein kinase phosphorylation status in head and neck squamous cell carcinoma.

BACKGROUND: To detect epigenetic changes in head and neck squamous cell carcinoma (HNSCC) and between metastatic and nonmetastatic tumors, we performed a systematic phosphorylation screening on different protein kinases. METHODS: The phosphorylation levels of the serine-threonine kinase Akt, of mitogen-activated protein kinase (MAPK), and of protein kinase C (PKC) beta and epsilon were measured in a series of 94 biopsy specimens, corresponding to 47 HNSCCs and paired controls taken from clinically uninvolved tissue of the same patients. RESULTS: Akt and MAPK were significantly underphosphorylated (two-sided p < .004) in tumors, whereas PKCs showed no differences from control samples.Second, although in control tissue there was a significant correlation between phosphorylation levels of Akt, MAPK, and PKC (all two-sided p < .05), many correlated activations were lost in tumors and even more in lymph node-positive tumors. Finally, p44 MAPK and Akt pThr308 were phosphorylated in a coordinated fashion only in lymph node-positive tumors (two-sided p < .01). CONCLUSIONS: This novel evidence documents important changes in the phosphorylation program during cancer progression of HNSCC.

Expression profiles of craniosynostosis-derived fibroblasts.

BACKGROUND: Craniosynostosis syndromes, a group of connective disorders characterized by abnormalities in vault osteogenesis and premature fusion of bone sutures, are associated with point mutations in FGF receptor family members. The cellular phenotype is characterized by abnormal extracellular matrix turnover. MATERIAL AND METHODS: We used primary cultures of periosteal fibroblasts derived from two different craniosynostosis syndromes, the Apert and Crouzon syndromes. The FGFR2 third immunoglobulin-like domain and its flanking linker regions were analyzed for mutation. DNA microarrays containing 19,200 cDNAs were used to study the gene expression profiles of Apert and Crouzon fibroblasts. The pathologic cells were compared to wild-type human periosteal fibroblasts. RESULTS: The P253R missense mutation and the G338R mutation were observed in Apert and Crouzon fibroblasts, respectively. The genetic profiles, as evaluated by DNA microarrays, yielded different clusters of expressed sequence tag (ESTs) expression within the experiment. Expression profiles from craniosynostosis-derived fibroblasts differ from those of wild-type fibroblasts (288 human ESTs, p< 0.01, pFDR = 0.12). Furthermore, two ESTs clusters discriminate the Crouzon from Apert fibroblasts. The differentially expressed genes cover a broad range of functional activities, including (1) bone differentiation, (2) cell-cycle regulation, (3) apoptotic stimulation, and (4) signaling transduction, cytoskeleton, and vesicular transport. CONCLUSIONS: The transcriptional program of craniosynostosis fibroblasts differs from that of wild-type fibroblasts. Expression profiles of Crouzon and Apert fibroblasts can also be distinguished by two EST expression clusters, thus hinting at a different genetic background.