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1.
BMC Cancer ; 8: 172, 2008 Jun 13.
Article in English | MEDLINE | ID: mdl-18554396

ABSTRACT

BACKGROUND: S100A4 is a metastasis-associated protein which has been linked to multiple cellular events, and has been identified extracellularly, in the cytoplasm and in the nucleus of tumor cells; however, the biological implications of subcellular location are unknown. Associations between a variety of posttranslational protein modifications and altered biological functions of proteins are becoming increasingly evident. Identification and characterization of posttranslationally modified S100A4 variants could thus contribute to elucidating the mechanisms for the many cellular functions that have been reported for this protein, and might eventually lead to the identification of novel drugable targets. METHODS: S100A4 was immuoprecipitated from a panel of in vitro and in vivo sources using a monoclonal antibody and the samples were separated by 2D-PAGE. Gels were analyzed by western blot and silver staining, and subsequently, several of the observed spots were identified as S100A4 by the use of MALDI-TOF and MALDI-TOF/TOF. RESULTS: A characteristic pattern of spots was observed when S100A4 was separated by 2D-PAGE suggesting the presence of at least three charge variants. These charge variants were verified as S100A4 both by western immunoblotting and mass spectrometry, and almost identical patterns were observed in samples from different tissues and subcellular compartments. Interestingly, recombinant S100A4 displayed a similar pattern on 2D-PAGE, but with different quantitative distribution between the observed spots. CONCLUSION: Endogenously expressed S100A4 were shown to exist in several charge variants, which indicates the presence of posttranslational modifications altering the net charge of the protein. The different variants were present in all subcellular compartments and tissues/cell lines examined, suggesting that the described charge variants is a universal phenomenon, and cannot explain the localization of S100A4 in different subcellular compartments. However, the identity of the specific posttranslational modification and its potential contribution to the many reported biological events induced by S100A4, are subject to further studies.


Subject(s)
Colorectal Neoplasms/chemistry , Colorectal Neoplasms/metabolism , Protein Processing, Post-Translational , S100 Proteins/isolation & purification , S100 Proteins/metabolism , Amino Acid Sequence , Blotting, Western , Colorectal Neoplasms/pathology , Electrophoresis, Gel, Two-Dimensional , HCT116 Cells , Humans , Immunoprecipitation , Molecular Sequence Data , Neoplasm Metastasis , Protein Isoforms , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , S100 Calcium-Binding Protein A4 , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
2.
Anticancer Res ; 27(3A): 1301-7, 2007.
Article in English | MEDLINE | ID: mdl-17593623

ABSTRACT

BACKGROUND: Melanoma is an aggressive disease that spreads quickly and is resistant to most therapeutic agents. In an effort to provide insight into the molecular basis of melanoma progression, the expression of 94 genes in 20 metastatic melanomas using a high-throughput real-time quantitative RT-PCR assay was analysed. MATERIALS AND METHODS: A TaqMan low density array (LDA) was designed containing probes/primers directed towards a cohort of genes previously found to be differentially expressed in an isogenic cell line model of melanoma progression. For each sample, cDNA was prepared and added to the quantitative assay. The resulting data were then analysed for correlations with clinical data. RESULTS: Clustering analysis divided the melanomas into two major subgroups based on gene expression patterns. When analysed individually, several genes were associated with overall survival, depth and type of the primary tumour. CONCLUSION: We have identified a selection of genes linked to melanoma progression and patient outcome.


Subject(s)
Melanoma/genetics , Melanoma/pathology , Disease Progression , Gene Expression Profiling , Humans , Reverse Transcriptase Polymerase Chain Reaction/methods
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