BMC Cancer 2008, 8:19 http://www.biomedcentral.com/1471-2407/8/19 apoptosis induction in tumors [43]. By Northern blot (Fig. 3A), we detected DCN mRNA only in the WM1552C cells and at very high levels, indicating that these libraries represent genes highly differentially expressed. ALS2CR7, a candidate gene of amyotrophic lateral sclerosis 2, encodes a putative protein kinase, based on Gene Ontol- ogy prediction, with no characterized function. As shown in Fig. 3B, although some signal for ALS2CR7 mRNA expression was detected in all eight cell lines analyzed, all four metastatic cell lines presented equivalently low sig- nals and the highest levels were detected in WM1552C, confirming the differential expression of this gene identi- fied in the RGP SSH library. The MBOAT1 gene encodes a hypothetical transmembrane protein containing an O- acyltransferase domain, also with no characterized func- tion and as predicted by its occurrence in the RGP library, we confirmed that its mRNA expression is higher in WM1552C (Fig. 3C). On the other hand, when a fragment of a gene identified in both libraries, YWHAZ (14.3.3ζ) was used as probe for Northern blot hybridization (Fig. 3D), we detected average signals of similar intensity between the RGP WM1552C and the metastatic cell lines, although some variation in the expression of this gene can be noted among the cell lines analyzed. From the Met library, we selected the genes MITF (repre- sented by one clone), PLP1 (24 clones) and HLA-DRA (55 clones). MITF is a transcription factor involved in melano- cyte differentiation and survival and even though it is rep- resented by only one sequence in the Met library, its differential expression between the RGP WM1552C and the metastatic cell lines was confirmed by Northern blot (Fig. 3E). High levels of MITF mRNA were detected in three of the cell lines independent of the growth phase, the RGP WM35, the VGP WM902, and the metastatic WM1617, but no signal was detected in the RGP WM1552C cells. PLP1 encodes a proteolipid protein involved in myelinization and as predicted by its presence in the Met library, the RGP WM1552C cell line showed no detectable expression of PLP1 mRNA while high levels were detected in the metastatic cell lines (Fig. 3F). To val- idate the expression pattern of the MHC class II HLA-DRA, the most redundant gene found in the Met library, we per- formed RT-PCR using cDNA from 6 cell lines and ana- lyzed the amplified product at 25, 28, 30 and 32 amplification cycles on agarose gel (Fig. 3G). The data confirmed high expression levels of HLA-DRA mRNA in the WM1617 metastatic cell line and a weak expression in the RGP cell line WM1552C (Fig. 3G). Moderate expres- sion levels were detected in cell lines of different growth phases (WM35, WM793 and WM852) and, interestingly, 1205Lu, which was selected in immunodefficient mouse from WM793, shows low expression levels. Also, high lev- els of the HLA-DRA protein were confirmed by flow cytometry for WM1617 and WM9 (data not shown). Therefore, the melanoma cell lines analyzed here express differential levels of HLA-DRA but without showing corre- lation to any particular phase of the tumor development. In summary, we conclude that all of the genes selected for validation confirmed the expression pattern predicted by their presence in only one of the two libraries. Genes of specific biological processes and from distinct chromosome locations are differentially enriched between the RGP and Met libraries In order to verify if specific classes of proteins are differen- tially enriched in the RGP or Met libraries, we submitted the two total lists of genes identified in our SSH libraries to a functional annotation based on the Gene Ontology, according to the biological processes. The annotation was performed using the software GOTM (Gene Ontology Tree Machine) that also compare the frequency of genes in each functional class with the expected frequency based on the distribution of all human genes throughout the GO functional classes. The GO functional classes that are significantly enriched in the RGP and Met libraries, in comparison to the distribution of all predicted human genes, are listed in Tables 2 and 3. Genes corresponding to proteins involved in nucleic acid binding are enriched in both libraries, however the number of genes and proc- esses related to this function is greater in the RGP library. In the Met library, regulation of transcription and RNA processing are the two processes involving nucleic acid binding proteins that were considered enriched. In the RGP library, we detected a large number of genes related to DNA metabolism, DNA repair, chromatin remodeling and RNA processing. In addition, proteins involved in cytoskeleton processes related to subcellular transport and localization, as well as proteins involved in macromole- cule degradation are also enriched in the RGP library. On the other hand, processes related to cell adhesion and cell migration were considered specifically enriched in the Met library. These processes include genes coding for components of extracellular matrix and several types of receptors such as G protein-coupled receptors, tyrosine kinase receptors, integrins and nuclear receptors. We also analyzed the chromosome location of all genes/ ESTs identified in the SSH libraries (Fig. 4). Interestingly, genes mapping to chromosome 1 are much more repre- sented in the RGP library (19%) than in the MET library (4%). Also, at lower extent, chromosomes 2, 6 and 12 had more genes identified in the RGP than in the Met library, whereas genes from chromosomes 11 and 13 showed an inverted pattern. Page 7 of 18 (page number not for citation purposes)