BMC Cancer 2008, 8:19 http://www.biomedcentral.com/1471-2407/8/19 [78], which is also implicated in the regulation of MITF [79], the other validated gene. genes with no characterized function or association to cancer, such as C18orf19. MITF encodes a transcription factor required for melano- cyte differentiation and survival [80,81] and we con- firmed high expression levels of MITF mRNA in one of the metastatic cell lines, WM1617, which also showed the highest level of PLP1 expression. The high levels of MITF in WM1617 cells must explain the occurrence of many melanocytic markers (known MITF targets) in the Met library, as TYRP1, TYR, DCT, MLANA. However, differ- ently from PLP1, high levels of MITF mRNA expression were not detected in the other three metastatic cell lines, but rather in cell lines from RGP and VGP tumor stages (WM35 and WM902). Consistent with this result, micro- array studies revealed MITF overexpression in a subset of primary and metastatic melanoma samples [16,77] and led to the proposal [77] of a classification of melanoma cell lines independently of tumor stage. Interestingly, in the latter study, PLP1, MITF and the melanocytic markers were all detected as co-regulated genes associated to high proliferation and low metastatic potential in groups of cell lines that included WM1617 and WM35. So it is likely that a set of the genes detected in the Met library is more importantly linked to tumor proliferation/survival than invasion. Although MITF is able to induce cell cycle arrest in melanocytes and melanoma cells in a p16 and p21 dependent manner [82,83], MITF gene was found to be amplified in melanoma and its overexpression induced transformation and drug resistance in BRAF mutant melanocytes [84]. In addition, recent works have identi- fied as MITF transcriptional targets genes such as the CDK2 [85], the hipoxia induced factor HIF1A [86] and the hepatocyte growth factor receptor MET [87], all of them presenting functions that contribute to tumor develop- ment. Thus, the cellular circuits in which MITF is engaged are clearly complex and when and how MITF contributes to melanoma development are open questions. Since many of the known MITF targets are represented in the Met library, this collection may contain novel MITF tar- gets and co-regulated genes whose identification will probably contribute to shed light on the MITF participa- tion in melanoma development. Other genes with consistent overexpression in tumor sam- ples in comparison to nevi and skin (Fig. 5C) compatible with an oncogenic role include genes already associated to melanoma progression, such as FN1, which codes for the matrix protein fibronectin [88]; genes with proposed roles in tumor growth and metastasis, such as ADAM10, which encodes a putative desintegrin metallopeptidase [89], SGK, which encodes a glucocorticoid regulated kinase [90], NRP2, which encodes neuropilin 2[91], and also When we looked for the expression profile for RGP and Met genes in the microarray data obtained by Hoek et al [92], 13 and 19 genes, respectively, were found amongst the genes up-regulated in melanoma cell lines as com- pared to melanocytes (Additional File 5, Tables S5 and S6). Eleven genes (Additional File 6, Table S7) from our subtractive libraries were also detected as differentially expressed between murine tumorigenic melanoma cells and a parental nontumoral cell line in a study using pro- teomics and SAGE analysis [93]. Up-regulation of one of these genes, NPM1 (nucleophosmin), was found at both protein and transcript levels in melanoma cells, compati- ble with the detection of this gene in our Met library. An accumulation of a specific form of the nucleophosmin protein was also detected in human melanoma cell lines (including several of the WM cells used here) in compari- son to normal melanocytes in another proteomics study [94]. Conclusion Altogether the data shown here strengthen previous evi- dence for several genes as candidate markers for melanoma progression and suggest that the subtractive libraries described are enriched in cancer-related genes, representing validated tools to be used in future studies for the identification of novel genes or pathways involved in melanoma progression. Competing interests The author(s) declare that they have no competing inter- ests. Authors' contributions JFS designed the study, constructed, and sequenced the SSH libraries, conducted all expression validation and comparisons, and drafted the manuscript. EME conceived the study, participated in its design and coordination and also drafted and revised the manuscript. Both authors read and approved the final manuscript. Additional material Additional file 1 Representative image of agarose gels containing PCR-amplified inserts from 96 randomly selected clones from the subtractive libraries. The images show that most clones from both RGP and Met libraries carry inserts and most of them are ≥ 600 bp in length. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2407-8-19-S1.pdf] Page 14 of 18 (page number not for citation purposes)