Quantitative Proteomic Studies of gamma-Secretase Inhibition in Hodgkin Lymphoma Cells Reveal Novel Insights into Notch Signaling.

Notch signaling has been implicated in the regulation of Hodgkin lymphoma (HL) survival via NF-kappaB. Notch signaling is dependent on the interaction of ligands with the transmembrane notch receptor. Ligand binding triggers proteolytic cleavage of the intracellular notch domain with subsequent translocation to the nucleus and activation of transcription factors. Gamma-secretase which catalyzes the proteolytic cleavage and release of the notch intracellular domain is critical in the mediation of notch signaling. Inhibition of gamma-secretase using 7{N-[N-(3,5-difluorophenyl)-L-alanyl]-s-phenyl-glycine t-butyl ester} (DAPT) in rat fetal thymocytes significantly reduces the expression of notch target genes. We identified proteins released by HL-derived cells into conditioned media including multiple upstream and downstream components of the notch signaling cascade, specifically: notch1, notch2, jagged1, jagged2, HES2, Hes4, GATA2 and GATA5. A proteomic analysis of the differentially expressed proteins among DAPT treated and untreated cells will reveal potential novel downstream mediators of notch signaling, increasing our understanding of HL pathogenesis. We sought to identify the proteomic consequences of notch signaling inhibition in L428 HL cells using a mass spectrometry-based proteomic approach. Treatment of L428 HL cells with DAPT (50μM) resulted in decreased cell proliferation as measured by the MTT assay which was associated with induction of p27Kip1. We utilized an endoproteinase catalyzed O16/O18 differential isotopic strategy to quantitatively determine the global proteomic changes following inhibition of the notch signaling pathway using DAPT. Proteins were collected from the cell lysate of treated and non-treated L428 cells, subjected to O16/O18 labeling and then analyzed by reverse-phase liquid chromatography coupled with electrospray ionization tandem mass spectrometry. A total of 156 proteins with 2 or more unique peptides were identified as being differentially expressed between treated and non-treated L428 cells. Proteins of diverse location and function were identified. Importantly a large number of proteins involved in transcription (12%; RelB, TRRAP, RB-associated protein, NCOR1), and located in the nucleus (27%; H2AO, FUSE binding protein 1, ANC5, SMYD1) were identified. Other important functional categories of the identified proteins included signaling activity (28%), and catalytic activity (41%). Several known proteins regulated by notch and involved with the regulation of notch activity such as (Histone acetyltransferase PCAF, RelB, N-COR1) were identified and found to be under expressed in treated cells. In addition, novel proteins with transcriptional and cell signaling activities have been identified, representing unique pathways that may be directly or indirectly affected by notch signaling. Our study represents the first comprehensive analysis of differentially expressed proteins following the inhibition of notch signaling. These results provide novel insights into our understanding of the pathogenesis and the role of notch signaling in HL