Analysis of candidate genes through a proteomics-based approach in primary cell lines from malignant melanomas and their metastases.
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.
Carta
Franco
F
Porto Conte Ricerche, 07041 Alghero, SS, Italy.
Demuro
Pietro P
PP
Zanini
Cristina
C
Santona
Antonella
A
Castiglia
Daniele
D
D'Atri
Stefania
S
Ascierto
Paolo A
PA
Napolitano
Maria
M
Cossu
Antonio
A
Tadolini
Bruna
B
Turrini
Franco
F
Manca
Antonella
A
Sini
Maria C
MC
Palmieri
Giuseppe
G
Rozzo
And Carla
AC
D018834:Chaperonin 60 / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional; D050883:HSC70 Heat-Shock Proteins / Q000032:analysis; D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000032:analysis; D006801:Humans; D008544:Melanocytes / Q000378:metabolism; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism; D018832:Molecular Chaperones; D009361:Neoplasm Invasiveness / Q000235:genetics*; D009363:Neoplasm Proteins / Q000032:analysis; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D018834:Chaperonin 60; D050883:HSC70 Heat-Shock Proteins; D055551:HSP27 Heat-Shock Proteins; C494371:HSPA8 protein, human; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D011506:Proteins