Analysis of Ras-induced oncogenic transformation of NIH-3T3 cells using differential-display 2-DE proteomics.
Ras proteins control at least three crucial signalling networks responsible for several cellular processes including anchorage independence, survival, and proliferation. Point mutations in one of the three ras genes are frequent in human tumours. In these tumours, Ras oncoproteins contribute significantly to the malignant phenotype, including deregulation of tumour-cell growth, apoptosis and invasiveness, and the ability to induce angiogenesis. Although significant strides have been made in understanding Ras biology, the collaborative actions of Ras effectors are still poorly understood. Here, we describe a proteomics approach to study global changes in protein expression in Ras-transformed NIH3T3 cells. We exploited 2-D difference gel electrophoresis (DIGE) for pre-separation fluorescent protein labelling with three separate dyes to reduce gel-to-gel variability, to increase sensitivity and dynamic range of protein detection, and to enhance quantification of dysregulated proteins. Proteins dysregulated (> 1.5-fold) by oncogenic Ras transformation reported to be implicated in Ras-regulated pathways include S-methyl-5-thioadenosine phosphorylase, stress-induced-phosphoprotein 1, galectin-1, annexin A7 (synexin), 60S acidic ribosomal protein P0, serine/threonine protein phosphatase type 1 (PP1alpha) and prohibitin. Significantly, we report for the first time the expression of the newly discovered cytokine IL-25 (or IL-17E) in mouse embryonic fibroblast cells and its down-regulation (2.1-fold) upon Ras-induced oncogenic transformation.
Ji
Hong
H
Joint Proteomics Laboratory, Ludwig Institute for Cancer Research and the Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.
Moritz
Robert L
RL
Kim
Yu-Sam
YS
Zhu
Hong-Jian
HJ
Simpson
Richard J
RJ
D016475:3T3 Cells / Q000378:metabolism*; D000818:Animals; D017310:Annexin A7 / Q000378:metabolism; D002460:Cell Line; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic / Q000378:metabolism*; D003626:Data Display; D015536:Down-Regulation; D015180:Electrophoresis, Gel, Two-Dimensional; D005456:Fluorescent Dyes; D037161:Galectins / Q000378:metabolism; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D011905:Genes, ras / Q000235:genetics*; D020381:Interleukin-17 / Q000378:metabolism; D051379:Mice; D009363:Neoplasm Proteins / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D000091402:Prohibitins; D040901:Proteomics / Q000295:instrumentation / Q000379:methods; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation
D017310:Annexin A7; D005456:Fluorescent Dyes; D037161:Galectins; D020381:Interleukin-17; D009363:Neoplasm Proteins; D000091402:Prohibitins; D012097:Repressor Proteins; D010749:Phosphoprotein Phosphatases; D016283:Proto-Oncogene Proteins p21(ras)