Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets

<p>Abstract</p> <p>Background</p> <p>Melanoma progression occurs through three major stages: radial growth phase (RGP), confined to the epidermis; vertical growth phase (VGP), when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH) to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor <it>KISS1</it>, lack of αvβ3-integrin and low levels of <it>RHOC</it>.</p> <p>Methods</p> <p>Two subtracted cDNA collections were obtained, one (RGP library) by subtracting the RGP cell line (WM1552C) cDNA from a cDNA pool from four metastatic cell lines (WM9, WM852, 1205Lu and WM1617), and the other (Met library) by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified.</p> <p>Results</p> <p>We identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library.</p> <p>Conclusion</p> <p>This study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in available expression study databases allowed us to point to a great potential of involvement in tumor progression for several of the genes identified here. A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts.</p

The use of Open Reading frame ESTs (ORESTES) for analysis of the honey bee transcriptome

BACKGROUND: The ongoing efforts to sequence the honey bee genome require additional initiatives to define its transcriptome. Towards this end, we employed the Open Reading frame ESTs (ORESTES) strategy to generate profiles for the life cycle of Apis mellifera workers. RESULTS: Of the 5,021 ORESTES, 35.2% matched with previously deposited Apis ESTs. The analysis of the remaining sequences defined a set of putative orthologs whose majority had their best-match hits with Anopheles and Drosophila genes. CAP3 assembly of the Apis ORESTES with the already existing 15,500 Apis ESTs generated 3,408 contigs. BLASTX comparison of these contigs with protein sets of organisms representing distinct phylogenetic clades revealed a total of 1,629 contigs that Apis mellifera shares with different taxa. Most (41%) represent genes that are in common to all taxa, another 21% are shared between metazoans (Bilateria), and 16% are shared only within the Insecta clade. A set of 23 putative genes presented a best match with human genes, many of which encode factors related to cell signaling/signal transduction. 1,779 contigs (52%) did not match any known sequence. Applying a correction factor deduced from a parallel analysis performed with Drosophila melanogaster ORESTES, we estimate that approximately half of these no-match ESTs contigs (22%) should represent Apis-specific genes. CONCLUSIONS: The versatile and cost-efficient ORESTES approach produced minilibraries for honey bee life cycle stages. Such information on central gene regions contributes to genome annotation and also lends itself to cross-transcriptome comparisons to reveal evolutionary trends in insect genomes

Novel Primate-Specific Genes, RMEL 1, 2 and 3, with Highly Restricted Expression in Melanoma, Assessed by New Data Mining Tool

Melanoma is a highly aggressive and therapy resistant tumor for which the identification of specific markers and therapeutic targets is highly desirable. We describe here the development and use of a bioinformatic pipeline tool, made publicly available under the name of EST2TSE, for the in silico detection of candidate genes with tissue-specific expression. Using this tool we mined the human EST (Expressed Sequence Tag) database for sequences derived exclusively from melanoma. We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression. Using a diverse panel of human tissues and cell lines, we validated the expression of a subset of three previously uncharacterized genes (clusters Hs.295012, Hs.518391, and Hs.559350) to be highly restricted to melanoma/melanocytes and named them RMEL1, 2 and 3, respectively. Expression analysis in nevi, primary melanomas, and metastatic melanomas revealed RMEL1 as a novel melanocytic lineage-specific gene up-regulated during melanoma development. RMEL2 expression was restricted to melanoma tissues and glioblastoma. RMEL3 showed strong up-regulation in nevi and was lost in metastatic tumors. Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes. The three genes are composed of multiple exons and map to 2q12.2, 1q25.3, and 5q11.2, respectively. They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found. Hairpin RNA secondary structures were also predicted. Concluding, this work offers new melanoma-specific genes for future validation as prognostic markers or as targets for the development of therapeutic strategies to treat melanoma

Identification of unannotated exons of low abundance transcripts in Drosophila melanogaster and cloning of a new serine protease gene upregulated upon injury

<p>Abstract</p> <p>Background</p> <p>The sequencing of the <it>D.melanogaster </it>genome revealed an unexpected small number of genes (~ 14,000) indicating that mechanisms acting on generation of transcript diversity must have played a major role in the evolution of complex metazoans. Among the most extensively used mechanisms that accounts for this diversity is alternative splicing. It is estimated that over 40% of <it>Drosophila </it>protein-coding genes contain one or more alternative exons. A recent transcription map of the <it>Drosophila </it>embryogenesis indicates that 30% of the transcribed regions are unannotated, and that 1/3 of this is estimated as missed or alternative exons of previously characterized protein-coding genes. Therefore, the identification of the variety of expressed transcripts depends on experimental data for its final validation and is continuously being performed using different approaches. We applied the Open Reading Frame Expressed Sequence Tags (ORESTES) methodology, which is capable of generating cDNA data from the central portion of rare transcripts, in order to investigate the presence of hitherto unnanotated regions of <it>Drosophila </it>transcriptome.</p> <p>Results</p> <p>Bioinformatic analysis of 1,303 <it>Drosophila </it>ORESTES clusters identified 68 sequences derived from unannotated regions in the current <it>Drosophila </it>genome version (4.3). Of these, a set of 38 was analysed by polyA⁺northern blot hybridization, validating 17 (50%) new exons of low abundance transcripts. For one of these ESTs, we obtained the cDNA encompassing the complete coding sequence of a new serine protease, named SP212. The <it>SP212 </it>gene is part of a serine protease gene cluster located in the chromosome region 88A12-B1. This cluster includes the predicted genes CG9631, CG9649 and CG31326, which were previously identified as up-regulated after immune challenges in genomic-scale microarray analysis. In agreement with the proposal that this <it>locus </it>is co-regulated in response to microorganisms infection, we show here that SP212 is also up-regulated upon injury.</p> <p>Conclusion</p> <p>Using the ORESTES methodology we identified 17 novel exons from low abundance <it>Drosophila </it>transcripts, and through a PCR approach the complete CDS of one of these transcripts was defined. Our results show that the computational identification and manual inspection are not sufficient to annotate a genome in the absence of experimentally derived data.</p

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets-2

Decorin) (), () and () of the RGP library; (14-3-3 ξ) () identified in both libraries; and () and () from the Met library were isolated and used as probes for hybridization in Northern blots containing total RNA from the melanoma cell lines indicated above the panels – mean that the corresponding cell line was not included in the Northern blot, and were introduced to allow alignment among panels. Northern blots were prepared as described in Figure 1. () identified in the Met library was validated by RT-PCR. For RT-PCR, total RNA samples (2 μg) from the indicated cell lines were, after DNase treatment, submitted to reverse transcription with Superscript II (Invitrogen) using oligo dT as primer and the cDNA was used as template for PCR amplification with primers. After 25, 28, 30 and 32 amplification cycles, 5 μl aliquots were collected for agarose gel electrophoresis. As endogenous control, a pair of primers for the (β-actin) mRNA was used. : Control RT-PCR amplification using as template RNA (DNase treated) without prior reverse transcription.<p><b>Copyright information:</b></p><p>Taken from "Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets"</p><p>http://www.biomedcentral.com/1471-2407/8/19</p><p>BMC Cancer 2008;8():19-19.</p><p>Published online 22 Jan 2008</p><p>PMCID:PMC2267200.</p><p></p

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets-4

E expression data for each gene group were submitted to SAM (FDR = 0) in a two-class analysis for detection of genes differentially expressed between primary and metastatic tumors and between non-neoplastic (skin and melanocytic nevi) and neoplastic (primary and metastatic melanomas) samples. The results from SAM analysis were extracted using SAMTERS and visualized by CLUSTER 3.0 and Java TreeView – Red and green squares represent genes up-regulated and down-regulated, respectively. (A) Expression profiles from primary and metastatic tumors for genes from the RGP library. (B – C) Expression profiles from non-neoplastic and neoplastic samples for genes from the RGP (B) and Met (C) libraries. Vertical blue lines on the left side indicate: (B) Genes from the RGP library that showed up-regulation in non-neoplastic samples; and (C) Genes from the Met library up-regulated in neoplastic tissues.<p><b>Copyright information:</b></p><p>Taken from "Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets"</p><p>http://www.biomedcentral.com/1471-2407/8/19</p><p>BMC Cancer 2008;8():19-19.</p><p>Published online 22 Jan 2008</p><p>PMCID:PMC2267200.</p><p></p

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets-3

total number of genes per human chromosome for each library; (B and C) Represent the chromosome locations for all genes identified in the RGP (B) and Met (C) libraries, along the length (bp) of all human chromosomes. The absence of genes mapping to Y chromosome in the RGP library is not explained by lack of this chromosome since the RGP cell line WM1552C was obtained from a male patient.<p><b>Copyright information:</b></p><p>Taken from "Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets"</p><p>http://www.biomedcentral.com/1471-2407/8/19</p><p>BMC Cancer 2008;8():19-19.</p><p>Published online 22 Jan 2008</p><p>PMCID:PMC2267200.</p><p></p

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets-0

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 transferred 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.<p><b>Copyright information:</b></p><p>Taken from "Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets"</p><p>http://www.biomedcentral.com/1471-2407/8/19</p><p>BMC Cancer 2008;8():19-19.</p><p>Published online 22 Jan 2008</p><p>PMCID:PMC2267200.</p><p></p

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets-6

duplicate samples of subtracted (S1 and S2) or non-subtracted (NS) cDNA of the RGP (WM1552C) and the metastatic (a pool of WM9, WM852, 1205Lu and WM1617) cell lines. PCR-2 represents the PCR product generated after 10 amplification cycles by nested-PCR using a specific primer for each adaptor. Note the difference between the subtracted and non-subtracted profiles.<p><b>Copyright information:</b></p><p>Taken from "Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets"</p><p>http://www.biomedcentral.com/1471-2407/8/19</p><p>BMC Cancer 2008;8():19-19.</p><p>Published online 22 Jan 2008</p><p>PMCID:PMC2267200.</p><p></p