Use of representational difference analysis and cDNA arrays for transcriptional profiling of tumor tissue.

Representational difference analysis of cDNA (cDNA-RDA) was used for a comparison of the global transcript level of tumor of the larynx and the corresponding normal epithelial tissue toward the end of detecting differentially expressed genes. Overall, some 130 gene fragments were identified. By sequence analysis and homology comparison, they could be put into several groups related to (potential) functions. Apart from genes whose overexpression was most likely a result of tumor growth or dedifferentiation of epithelial tissue, a lot of genes were isolated that play major roles in signal transduction pathways or apoptosis or act as oncogenes or tumor suppressor genes, in addition to new, entirely unknown genes. Moreover, some cDNAs of known genes were identified that derived from unconventional splicing activity or other transcript modifications. All identified fragments were arrayed on solid support and used for reverse Northern blot analyses. The use of preselected RDA fragments as targets in array-based profiling experiments circumvents many of the problems encountered when dealing with large clone libraries.

Expression of ril, a novel LIM domain gene, is down-regulated in Hras-transformed cells and restored in phenotypic revertants.

Several candidate genes involved in the maintenance of normal growth control (H-rev) were identified by differential expression cloning on the assumption that they are expressed in phenotypically normal rat cells and repressed in closely related H-ras transformed cells. Previously the genes coding for lysyl oxidase (H-rev142) and for an 18K-protein of unknown function (H-rev107) were recovered as cDNAs by subtraction cloning. Here we describe the identification and expression pattern of ril, a novel member of the heterogeneous group of genes encoding proteins with LIM/double zinc finger domains. The ril gene is expressed in normal fibroblasts and down-regulated in H-ras-transformed derivatives. Expression is restored in several independent phenotypic revertants derived from H-ras transformed cells. The predicted protein product of ril harbors a single LIM domain but lacks a homeodomain. The ril gene is highly conserved during evolution and is transcribed in various normal cell lines. Northern blot analysis and in situ hybridization studies showed that ril is expressed in meiotic spermatocytes, in somites of developing mice, and in a wide variety of tissues of adult mice.