ORIGINAL ARTICLESAnalysis of candidate genes through a proteomics-based approach in primary cell lines from malignant melanomas and their metastasesCarta, Francoa; Demuro, Pietro P.b; Zanini, Cristinac; Santona, Antonellaa; Castiglia, Danieled; D'Atri, Stefaniad; Ascierto, Paolo A.e; Napolitano, Mariae; Cossu, Antoniof; Tadolini, Brunag; Turrini, Francoc; Manca, Antonellab; Sini, Maria C.b; Palmieri, Giuseppeb; Rozzo, and Carlabon behalf of the Italian Melanoma Intergroup (IMI)Author Information aPorto Conte Ricerche, 07041 Alghero (SS), Italy bIstituto di Chimica Biomolecolare-Sezione di Sassari, C.N.R., 07040 Sassari, Italy cCentro Ricerca Medicina Sperimentale, 10126 Torino, Italy dIstituto Dermopatico dell'Immacolata, 00167 Roma, Italy eIstituto Nazionale Tumori ‘Fondazione G. Pascale’, 80131 Napoli, Italy fIstituto di Anatomia Patologica gDipartimento Scienze Biomediche, Università di Sassari, 07100 Sassari, Italy Sponsorship: This work was supported by Ricerca Finalizzata Ministero della Salute (F.S.N.), Regione Autonoma della Sardegna, Fondazione Banco di Sardegna, Compagnia di San Paolo (Oncology Programme). Correspondence and requests for reprints to Giuseppe Palmieri, Istituto di Chimica Biomolecolare-Sezione di Sassari, C.N.R., Località Tramariglio, Alghero – 07040 Santa Maria La Palma (Sassari), Italy Tel: +39 079 396 1033; fax: +39 079 396 1036; e-mail: [email protected] Received 25 November 2004 Accepted (after revision) 16 March 2005 Melanoma Research: August 2005 - Volume 15 - Issue 4 - p 235-244 Buy Abstract Proteomics provides a powerful approach for screening alterations in protein expression and post-translational modification associated with particular human diseases. In this study, the analysis of protein expression was focused on malignant melanoma in order to determine the candidate genes involved in tumour progression. The proteomes of cultured melanocytes and of cell lines from primary and metastatic lesions of one malignant melanoma patient were profiled using two-dimensional electrophoresis (2-DE) and mass spectrometry. Differentially expressed proteins were confirmed by 2-DE and mass spectrometry on an additional four malignant melanoma cell lines. Total RNA from the first subset of cell lines was used for quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of the candidate genes identified after proteomics analysis. A very high similarity was observed in the 2-DE maps of two malignant melanoma cell lines derived from primary and secondary lesions of the same patient. Mass spectrometry identified 37 proteins which were found to be more abundant in tumour cells in comparison with control melanocytes (as confirmed on additional cell lines), with a relatively high prevalence of stress proteins. Eight candidate genes (PRDX2, HSP27, HSP60, HSPA8, HSP9B, STIP1, PDI and P4HB) were further characterized by evaluating their messenger RNA expression levels through real-time RT-PCR analysis. Overexpression of HSP27, HSP60 and HSPA8 and downregulation of PRDX2 were observed in cells from metastatic malignant melanoma in comparison with those from primary melanoma. Although further investigations with larger numbers of paired normal and tumour samples are needed, our findings strongly suggest that the dysregulation of stress pathways may be involved in melanoma progression. © 2005 Lippincott Williams & Wilkins, Inc.