Discovery of Biomarkers for Hodgkin Lymphoma Using Proteomics Technology.

Hodgkin lymphoma (HL) is characterized by a minority of neoplastic cells, the Hodgkin and Reed-Sternberg cells (HRS cells) and an inflammatory background. Abnormal cytokine and chemokine production may play an important role in the proliferation of HRS cells, the reactive background formation and the impaired immune response encountered in patients with HL. To detect new proteins that might be involved in the interaction between the HRS cells and the inflammatory background cells and that also might serve as useful biomarkers for HL, we used different proteomic techniques to study the proteome of HRS cells.

Methods: The first step is to determine the secretome of HRS cells in vitro. Therefore, the cells needed to be cultured without serum. Hodgkin cell lines L428 and L1236 were washed extensively to deplete serum proteins and cultured in RPMI media without serum for 6–72 hours. The supernatant was collected, concentrated, and analyzed by Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS) to determine the protein composition and as a quality check. After culture process optimization, sample fractionation was carried out by SDS-PAGE. The whole lane was excised in 30–35 bands and trypsine digestion was performed on every band. Peptide mixtures were analyzed by LC-MS/MS for identification of the proteins.

Results: We tested several culture approaches for the Hodgkin lymphoma cell lines for proteomics purpose. Serum-free media were found to be inappropriate because of the abundant presence of several proteins. We next tested efficiency of washing steps and culture characteristics of 5 Hodgkin lymphoma cell lines in RPMI without serum. Cell viability, TARC and MDC levels in the culture supernatant at different time points and also SELDI-TOF-MS spectra indicated that culturing for 6–24 hours gave the best results. For protein identification we started with analysis of small proteins from supernatant of the L428 cell line, which has the highest TARC production. Gel bands in the region between 6–14 kDa were isolated from SDS-PAGE and treated for protein identification. This revealed a total of 64 proteins identified with ≥95% confidence. Application of SecretomeP2.0 (Protein Eng.Des.Sel., 2004. TMHMM 2.0) software indicated the presence of 28 secreted proteins including TARC. A similar approach was used for the L1236 Hodgkin lymphoma cell line, but the whole mass range (3–188 kDa) was analyzed in this case. We identified a total of 215 different proteins, out of which 85 are secreted (24 proteins follow a classical pathway while the other 61 follow a non-classical pathway). The non-secreted proteins were localized to the ER lumen, mitochondrion, nucleus, and membrane. These proteins are probably derived from cells breaking up during culture or centrifugation.

Conclusion: In this study we identified 85 secreted proteins in L1236 supernatant, and 28 secreted proteins in the low molecular mass range (6–14kDa) in L428 supernatant. Some of the secreted proteins, including TARC, macrophage migration inhibitory factor (MIF), CD44 and Activated Leukocyte Cell Adhesion Molecule (ALCAM), are known to be involved in inflammatory process, lymphocytes homing or cell adhesion. The most consistently observed proteins will be tested for potential use as biomarkers in the sera of HL patients.