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Purpose:To better understand the molecular mechanisms of malignant melanoma progression and metastasis, gene expression profiling was done of primary melanomas and melanoma metastases. Experimental Design: Tumor cell^specific gene expression in 19 primary melanomas and 22 melanoma metastases was analyzed using oligonucleotide microarrays after laser-capture microdissection of melanoma cells. Statistical analysis was done by random permutation analysis and support vector machines. Microarray data were further validated by immunohistochemistry and immunoblotting. Results: Overall, 308 genes were identified that showed significant differential expression between primary melanomas and melanoma metastases (false discovery rate V 0.05). Significantly overrepresented gene ontology categories in the list of 308 genes were cell cycle regulation, mitosis, cell communication, and cell adhesion. Overall, 47 genes showed up-regulation in metastases. These included Cdc6, Cdk1, septin 6, mitosin, kinesin family member 2C, osteopontin, and fibronectin. Down-regulated genes included E-cadherin, fibroblast growth factor binding protein, and desmocollin 1 and desmocollin 3, stratifin/14-3-3r, and the chemokine CCL27. Using support vector machine analysis of gene expression data, a performance of >85% correct classifications for primary melanomas and metastases was reached. Further analysis showed that subtypes of primary melanomas displayed characteristic gene expression patterns, as do thin tumors (V1.0 mm Breslow thickness) compared with intermediate and thick tumors (>2.0 mm Breslow thickness). Conclusions: Taken together, this large-scale gene expression study of malignant melanoma identified molecular signatures related to metastasis, melanoma subtypes, and tumor thickness. These findings not only provide deeper insights into the pathogenesis of melanoma progression but may also guide future research on innovative treatments.The incidence of malignant melanoma is steadily increasing with a present lifetime risk of 1 in 75 among the Caucasian population (1). The underlying factors for this phenomenon are largely unknown. After diagnosis of malignant melanoma, the single most important factor for the prognosis of melanoma patients is vertical tumor thickness as described earlier by Breslow (2). It could be shown that tumors of a few-millimeter thickness already show a high potential for metastasis with a fatal outcome for the patient. In the metastatic stage, melanoma patients have only few treatment options, consisting of monochemotherapies with dacarbazine (DTIC) or temozolomide, or polychemotherapy regimens combining DTIC with other chemotherapeutic agents such as cisplatin and 1,3-bis(2-chloroethyl)-1-nitrosourea (3 -5). Although significant clinical response rates were achieved by these treatment modalities, there was no substantial effect on the overall survival of these patients. Unfortunately, little is known about factors that contribute to the process of melanoma progression and metastasis.In recent years, DN...

We examined the repertoire and extent of inflammation dependent gene regulation in a bovine mammary epithelial cell (MEC) model, to better understand the contribution of the MEC in the immune defence of the udder. We challenged primary cultures of MEC from cows with heat inactivated Escherichia coli pathogens and used Affymetrix DNA-microarrays to profile challenge related alterations in their transcriptome. Compared to acute mastitis, the most prominently activated genes comprise those encoding chemokines, interleukins, beta-defensins, serum amyloid A and haptoglobin. Hence, the MEC exert sentinel as well as effector functions of innate immune defence. E. coli stimulated a larger fraction of genes (30%) in the MEC belonging to the functional category Inflammatory Response than we recorded with the same microarrays during acute mastitis in the udder (17%). This observation underscores the exquisite immune capacity of MEC. To more closely examine the adequacy of immunological regulation in MEC, we compared the inflammation dependent regulation of factors contributing to the complement system between the udder versus the MEC. In the MEC we observed only up regulation of several complement factor-encoding genes. Mastitis, in contrast, in the udder strongly down regulates such genes encoding factors contributing to both, the classical pathway of complement activation and the Membrane Attack Complex, while the expression of factors contributing to the alternative pathway may be enhanced. This functionally polarized regulation of the complex complement pathway is not reflected in the MEC models.

IntroductionDiscrimination of rheumatoid arthritis (RA) patients from patients with other inflammatory or degenerative joint diseases or healthy individuals purely on the basis of genes differentially expressed in high-throughput data has proven very difficult. Thus, the present study sought to achieve such discrimination by employing a novel unbiased approach using rule-based classifiers.MethodsThree multi-center genome-wide transcriptomic data sets (Affymetrix HG-U133 A/B) from a total of 79 individuals, including 20 healthy controls (control group - CG), as well as 26 osteoarthritis (OA) and 33 RA patients, were used to infer rule-based classifiers to discriminate the disease groups. The rules were ranked with respect to Kiendl’s statistical relevance index, and the resulting rule set was optimized by pruning. The rule sets were inferred separately from data of one of three centers and applied to the two remaining centers for validation. All rules from the optimized rule sets of all centers were used to analyze their biological relevance applying the software Pathway Studio.ResultsThe optimized rule sets for the three centers contained a total of 29, 20, and 8 rules (including 10, 8, and 4 rules for ‘RA’), respectively. The mean sensitivity for the prediction of RA based on six center-to-center tests was 96% (range 90% to 100%), that for OA 86% (range 40% to 100%). The mean specificity for RA prediction was 94% (range 80% to 100%), that for OA 96% (range 83.3% to 100%). The average overall accuracy of the three different rule-based classifiers was 91% (range 80% to 100%). Unbiased analyses by Pathway Studio of the gene sets obtained by discrimination of RA from OA and CG with rule-based classifiers resulted in the identification of the pathogenetically and/or therapeutically relevant interferon-gamma and GM-CSF pathways.ConclusionFirst-time application of rule-based classifiers for the discrimination of RA resulted in high performance, with means for all assessment parameters close to or higher than 90%. In addition, this unbiased, new approach resulted in the identification not only of pathways known to be critical to RA, but also of novel molecules such as serine/threonine kinase 10.

Metastases are responsible for cancer deaths, but the molecular alterations leading to tumor progression are unclear. Overexpression of the E2F1 transcription factor is common in high-grade tumors that are associated with poor patient survival. To investigate the association of enhanced E2F1 activity with aggressive phenotype, we performed a gene-specific silencing approach in a metastatic melanoma model. Knockdown of endogenous E2F1 via E2F1 small hairpin RNA (shRNA) expression increased E-cadherin expression of metastatic SK-Mel-147 melanoma cells and reduced their invasive potential but not their proliferative activity. Although growth rates of SK-Mel-147 and SK-Mel-103 xenograft tumors expressing E2F1 shRNA or control shRNA were similar, mice implanted with cells expressing E2F1 shRNA had a smaller area of metastases per lung than control mice (n = 3 mice per group; 5% vs 46%, difference = 41%, 95% confidence interval = 15% to 67%; P = .01; one-way analysis of variance). We identified epidermal growth factor receptor as a direct target of E2F1 and demonstrated that inhibition of receptor signaling abrogates E2F1-induced invasiveness, emphasizing the importance of the E2F1-epidermal growth factor receptor interaction as a driving force in melanoma progression that may serve as a paradigm for E2F1-induced metastasis in other human cancers.

Interleukin 6 (IL-6) is a growth and survival factor for multiple myeloma cells. As we report here, the IL-6-dependent human myeloma cell line INA-6 responds with a remarkably rapid and complete apoptosis to cytokine withdrawal.

SSX genes show extensive nucleotide sequence conservation but little is known of their function. Disruption of SSX1 or SSX2, by chromosome translocation and`inframe' fusion to SYT, is a consistent feature of synovial sarcomas. The resulting SYT-SSX1/SSX2 proteins are activators of transcription; transactivation function is located in SYT. Unrearranged SSX1 can repress transcription, and this has been attributed to a putative KruÈ ppel associated box (KRAB) repression domain at the N-terminus. Here we isolated SSX-KRAB domains to speci®cally measure repression activity, using a previously characterized KOX1-KRAB domain as a control. In our repressor assay SSX1-and SSX2-KRAB domains down-modulated the transactivation of a reporter gene by threefold, compared with 83-fold repression achieved by KOX1-KRAB in the assay. Yeast two-hybrid analysis showed that SSX1-KRAB, unlike KOX1-KRAB, fails to interact with the KRAB corepressor TIF1b. These results raise questions about the evolutionary and functional relationship of SSX-KRAB and typical KRAB domains of KruÈ ppel zinc ®nger genes. We found that full-length SSX1 showed potent (74-fold) repression in our repressor assay, indicating the existence of a repression domain distinct from SSX-KRAB. By assaying deletion constructs of SSX1 we localized repression activity to 33 amino acids at the C-terminus. This novel domain is conserved between SSX family members, and, unlike the KRAB-related domain, is retained on fusion with SYT. This has important implications in understanding the mechanism by which the SYT-SSX fusion protein could contribute to neoplasia.Keywords: sarcoma; SSX; KRAB; transcription Chromosome translocation t(X;18)(p11.2;q11.2) (Clark et al., 1994;Crew et al., 1995) is a diagnostic feature of human synovial sarcomas, in some cases representing the sole cytogenetic abnormality (Sandberg and Bridge, 1994). All the available evidence indicates that this translocation is a key event in tumorigenesis. In all tumours characterized, the SYT gene on chromosome 18 is juxtaposed`in-frame' with either the SSX1 gene or SSX2 gene on chromosome X (Figure 1). SSX1 and SSX2 are now known to be members of a highly conserved multigene family Gure et al., 1997), and the SSX loci that have been mapped are all located in chromosome band Xp11.2 (Crew et al., 1995;. In contrast to SYT, which is a widely expressed gene (de Bruijn et al., 1996; J Knight., unpublished data), SSX transcripts show a very restricted distribution in adult human tissues. So far, SSX1 and SSX2 expression has only been detected in testis and thyroid (Crew et al., 1995;Tureci et al., 1996;Gure et al., 1997).As yet, little is known about the normal biological functions of the SYT and SSX gene products. No DNA binding sequences are recognizable in the SYT or SSX proteins. However, when coupled to a GAL4 DNA binding domain in in vitro reporter assays, SYT can activate transcription (70-fold activation) and SSX1 can repress transcription (50-fold repression) from a minimal promoter in NIH3T3 ®broblasts (Brett ...

A, Neidel J, Lehrach H, Thiesen HJ, Ruiz P, Bläß S. Molecular signatures and new candidates to target the pathogenesis of rheumatoid arthritis.