BMC Cancer 2008, 8:19 http://www.biomedcentral.com/1471-2407/8/19 aKFnIiSgdSum1reant1adstRaHticOCmemlaRnNomAaexceplrlelsinseiosn in a panel of RGP, VGP KISS1 and RHOC mRNA expression in a panel of RGP, VGP and metastatic melanoma cell lines. Com- parison of the expression levels of the KISS1 metastasis sup- pressor gene (A) and the small GTPase RHOC (B) among melanoma cell lines of different stages of tumor progression supported the selection of WM1552C cell line as the RGP representative for suppression subtractive hybridization against a pool of metastatic cell lines. Samples of 20 μg of total RNA from different melanoma cell lines were submitted to electrophoresis in 1% agarose-formaldehyde gel and trans- ferred to nylon membrane (Hybond N, Amersham Pharmacia Biotech) by standard methods. Fragments of the indicated genes were radiolabeled with [α-32P]-dCTP by random- priming (Rad-prime kit, Invitrogen) and used as probes for Northern blot hybridization. In order to correct for loading differences, after stripping, the blots were probed with a ACTB (β-actin) cDNA fragment. The RHOC mRNA was detected in all melanoma cell lines, presenting an elevated expression particularly in the RGP cell line WM1789 (Fig. 1B). Also, WM1552C was previ- ously shown to lack expression of αvβ3 integrin [8] in contrast to WM793 and the metastatic cell lines WM9 and 1205Lu [41]. Here, we confirmed by flow cytometry that WM1552C cells do not express αvβ3 integrin, while we observed expression of this integrin in WM35 (RGP), WM278 (VGP) and WM1617 metastatic cells (data not shown). In addition, we found that expression of these molecular markers was compatible with observations made in our laboratory (unpublished data) that WM1552C is more sensitive to apoptosis triggered by cell adhesion impairment (anoikis) than WM35 cells and that both WM35 and WM1789 cell lines were capable to gen- erate slowly growing tumor in SCID mice, in contrast with WM1552C that in a preliminary assay was unable to induce visible primary tumor when injected (2 × 106 cells) into SCID mice in the same conditions. Furthermore, spontaneous transformation towards a more malignant phenotype has been pointed out for WM35 cell line [7] and, indeed, in contrast to WM1552C, WM35 cells were recently shown to express the melanoma chondroitin sul- fate proteoglycan (MCSP), a surface molecule implicated in enhanced tumor migration, invasion and anchorage- independent survival [42]. In view of these contrasts, although WM1552C cells carry the BRAF mutation V599E, they appears to retain the phenotype of a less aggressive melanoma tumor as compared with the other cell lines of this collection and therefore it was the only cell line selected as representative of the RGP stage for this study. Two subtracted cDNA collections were obtained, one of cDNA from the RGP cell line WM1552C subtracted from a cDNA pool of four metastatic cell lines (WM9, WM852, 1205Lu and WM1617), which we named RGP library. The second library, referred as Met library, was obtained by the reverse subtraction. The cDNA profiles generated by the subtraction process are shown in Fig. 2. Cloning of these cDNAs into the pGEM vector allowed us to obtain 2016 clones for the RGP library and 1920 clones for the Met library. PCR analysis of 395 and 336 randomly selected clones from the RGP and Met libraries, respectively, indi- cated that 97% of the clones from the RGP library and 98% from the Met library contained inserts and that the insert size of most clones was ≥ 600 bp (images of repre- sentative agarose gels for each library are shown in the Additional File 1, Fig. S1). A total of 753 clones from both libraries were sequenced and annotated, as summarized in Table 1. The sequences were submitted to GenBank [GenBank accession num- bers: ES315683–ES316435]. Most sequences (94–95%) corresponded to annotated mRNA sequences. The sequence redundancy within each library is low, 288 dif- ferent genes are represented in the RGP library (non- redundancy of 82%) and 217 in the Met library (non- Page 5 of 18 (page number not for citation purposes)