Differentially Expressed Protein Patterns in Chronic Lymphocytic Leukemia (CLL) after Thymosin beta4 (Tb4) and Lenalidomide (Len) Treatment: Two-Dimensional Gel Electrophoresis (2DE) Analysis

Proteomic approaches are commonly secondary to genetic studies but are essential in the multi-disciplinary field of hematological research. As opposed to mRNA microarray data, proteomics provides a better understanding of which proteins are actually expressed, although the identification of specific proteins remains challenging (Unwin et al Blood Rev 2014; Boyd J Proteomics 2010). In neoplastic hematology such as CLL, protein studies have contributed to the elucidation of disease mechanisms, defined prognostic or therapeutic biomarkers (Boyd J Proteomics 2010). In this study we used proteomics and 2DE analysis to evaluate differential protein expression patterns after treatment with Len. Len can improve immune dysfunction in CLL by repairing F-actin polymerization and signaling at the immunological synapse (Ramsay et al 2008 J Clin Invest). Our previous data obtained from MALDI-TOF analysis identified Tβ4, a G-actin sequestering protein involved in the regeneration of injured tissues and cell migration, as a downregulated protein in CLL patients, also confirmed by an independent GEP analysis comparing B-cell from CLL cases (n=80) and normal controls (n=6), supported by Tβ4 mRNA down-regulation in CLL (3604±1244 vs 5715±1004, respectively; mean±SD; p=0.001). Here, we investigated whether purified B-CLL cells respond differently to the chemoattractant SDF1a and whether different protein expression patterns can be identified after exogenous Tβ4 and Len treatment using 2DE analysis.

Highly purified B-CLL lymphocytes were isolated from untreated Binet stage A CLL patients prospectively enrolled from diagnosis (O-CLL1 protocol, clinicaltrial.gov identifier NCT00917540) and healthy controls. Tb4 was identified by MALDI TOF using 100 patient samples. Next, cells were pre-treated with Len (5uM) and then treated with Tb4 (100nM) for 30min. Cells were plated in transwells using 5.0 um pores with SDF1a as chemoattractant for migration assays. Protein was extracted from CLL cell pellets by RIPA buffer and quantified. Sample preparation and 2DE was performed as described by Scielzo et al (J Clin Inves, 2005). Protein samples (100 ug) were applied to 7-cm IPG strips, pH 3–11NL (Amersham Biosciences), respectively, by in-gel rehydration. Isoelectric focusing was performed with a Protean i12 IEF system (Biorad). Strips were equilibrated and loaded onto 9–16% gradient acrylamide SDS-PAGE gels for the second dimension separation. Silver nitrate staining (Sinha P et al Proteomic, 2001) was used to visualize proteins and images were digitally acquired (ChemiDoc MP, Biorad) and spots were analyzed using PDQuest basic 2D Gel Analysis Software (Biorad).

CLL samples with the lowest Tβ4 expression (n=12) also had higher F-actin levels as evaluated by FC analysis than normal controls. B-CLL strongly responded to the migratory stimulus SDF-1a, which was further increased (by 20%) in presence of Len treatment, likely due to an alteration in actin remodeling and changes in the expression of unknown proteins. Purified CD19+CD5+ leukemic cells were lysed and proteins resolved on 2DE and visualized by silver nitrate staining. The protein profile analysis on silver-stained gels showed a number of protein spots ranging from 18 to 60kDa that were differentially expressed with respect to untreated cells. Our preliminary qualitative analysis suggests that there are groups of proteins with a lower expression in the presence of Tβ4 or Len, which are more strongly inhibited following exposure to their combination. Conversely, an opposite pattern of protein expression was observed whereby an additive effect on protein expression was observed by combined exposure to Tβ4 and Len. This approach allowed us to identify an altered protein expression pattern after treatment with Len and Tβ4, and may be useful to identify changes in expression profiles of CLL proteins, which may translate into functional differences in the malignant clone.