Cancer gene validation using ribozymes and macroarray.

Transcription factor AP-2 seems to play an important role in the malignancy of melanoma. In this study, we constructed ribozyme expression vectors to suppress the expression of AP-2 (AP-2-ribozymes) and then examined gene expression in human A375P melanoma cells that stably expressed ribozymes targeted to the AP-2 transcript. A comparison of the gene-expression profiles of A375P cells that expressed AP-2-ribozymes and those transfected with the empty vector revealed changes in levels of expression of several genes. Here we described that the combination of gene suppression by ribozymes and the analysis of gene expression using a macro-array provides a good approach for elucidating signal transduction pathways. These results provide further insight into the role of AP-2 in human melanoma cells.

Identification of AP-2-regulated genes by macroarray profiling of gene expression in human A375P melanoma.

Transcription factor AP-2 is a negative regulator of metastasis. Its expression is down regulated with progression of melanoma cells to metastasis. In this study, we performed macroarray profiling of gene expression of human A375P melanoma cells and their derivatives with overexpression of AP-2 and dominant-negative AP-2. Such comprehensive analysis lead to an identification of genes such as MMP-2, E-cadherin, melanoma adhesion molecule, early growth response 1, fibroblast growth factor 3, ubiquitin C, histone deacetylase 3 and integrin alpha 5,7, beta 3,5 as regulated by AP-2. Whereas some of these are known as AP-2-regulated genes, the others are not so far. Thus the study reports for the first time identification of new genes regulated by AP-2 that may be involved in metastasis of melanoma.

A functional gene discovery in cell differentiation by hybrid ribozyme and siRNA libraries.

Recently, we developed a gene discovery system that can identify functional genes using a randomized hybrid ribozyme library. In this system, inhibition of the expression of a particular gene by active ribozymes was reflected by a change in a particular phenotype, the method allowed the identification of functional genes. In the case of identification of functional genes for apoptosis pathways, we identified many pro-apoptotic genes in TNF-alpha and Fas-mediated apoptosis pathways. In this study, we tried to identify the functional genes that are necessary for the retinoic acid (RA)-induced cell differentiation using randomized ribozyme and siRNA libraries. We succeeded to identify the several differentiation factors. Therefore, our gene discovery system based on randomized ribozyme and siRNA libraries are high potential to identify the differentiation and undifferentiation factors in the post genome era.