Quantitative Proteomic and Transcriptional Analysis of the Response to the p38 Mitogen-activated Protein Kinase Inhibitor SB203580 in Transformed Follicular Lymphoma Cells

The p38 mitogen-activated protein kinase (MAPK) is a key mediator of stress, extracellular-, growth factor-, and cytokine-induced signaling, and has been implicated in the development of cancer. Our previous work showed evidence for p38 MAPK activation in a subset of transformed follicular lymphomas (Elenitoba-Johnson et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 7259). We demonstrated that inhibition of p38 MAPK by SB203580 resulted in doseand time-dependent caspase-3-mediated apoptosis. In order to further elucidate the basis of the cellular effects of SB203580, we have employed a systems biologic approach involving cDNA microarray and quantitative proteomic analysis of transformed follicular lymphoma derived-cells (OCI Ly-1) treated with SB203580. Gene expression profiling revealed differential expression (>1.5-fold) of 374 genes/ESTs in cells treated for 3 h and 515 genes/ESTs in cells treated for 21 h. The majority (52% at 3 h and 91% at 21 h) were down-regulated, including genes encoding growth cytokines, transcriptional regulators and cytoskeletal proteins. Quantitative proteomic analysis using ICAT-LC-MS/MS identified 277 differentially expressed proteins at 3 h and 350 proteins at 21 h of treatment with SB203580, the majority of which were also downregulated. Analysis of functional groups of the differentially expressed proteins implicated components of diverse overlapping pathways including the IL-6/phosphatidylinositol 3-kinase, insulin-like growth factor 2/Ras/Raf, WNT8d/Frizzled, MAPK-activated protein kinase 2, and nuclear factor B. The differential phosphorylation status of selected kinase-active proteins was validated by Western blotting analysis. Our complementary genomic and proteomic approach reveal the global cellular consequences of SB203580 treatment and provide insights into its growth inhibitory effect on transformed follicular lymphoma cells. Molecular & Cellular Proteomics 3:820–833, 2004. An important aim of functional genomic research is to globally identify and quantify specific proteomic and/or transcriptomic changes that are associated with physiologic or diseased states, thus enabling the elucidation of genes/proteins and signaling pathways that are associated with the phenotypic expression of a particular cellular state. The currently available microarray technologies permit the quantification of tens of thousands of gene transcripts and have been largely successful in demonstrating the global transcriptional changes accompanying the transition between cellular quiescence and activation, or during transition from normal to pathologic states (1–7). Proteomics, on the other hand, allows identification and quantification of up to a few thousand proteins, limited by the currently available technologies and the complex nature of the proteome especially in higher eukaryotes and mammalian cell systems. While the most widely used proteomics approach is the two-dimensional (2-D) gel-based method followed by MS (8–10), the recent development of multidimensional liquid chromatographic methods combined with MS/MS (LC-LC-MS/MS) has permitted sensitive detection of low-abundance proteins, membrane proteins and proteins with extreme pI (11–15). The ability to perform global quantitative proteomics has been significantly enhanced by the advent of the ICAT-based technology that is efficient in simplifying the proteome, and in combination with threedimensional 3-D LC-MS/MS permits detection and quantifi-