Abstract
* A.R. and M.L. have contributed equally.
INTRODUCTION: Myelodysplastic syndrome (MDS) is a clonal disease that arises from the expansion of mutated hematopoietic stem cells. Oxidative stress could play an important role in the pathology of MDS since a correlation has been observed between high ROS levels and decreased survival in MDS patients. In addition, the oxidation of proteins leads to their aggregation, change and conformational changes that can cause the loss of their functions. Due to alteration of the proteome has been described in patients with MDS, in this study we considered the discovery of protein carbonylation to analyze its oxidation pattern. In addition, as the oxidative stress signaling pathways can regulate the cell cycle through p53, we analyzed p21, an important p53 target during the stress response. Finally, the possible benefit of treatment with Deferasirox as antioxidant therapy was studied.
METHODS: Carbonylation pattern was analyzed in the different cell lines (n = 14, 6 MDS, 4 reactive controls and 4 MDS + DFX) by immunohistochemistry by derivatization with 2,4-dinitrophenylhydrazine (DNPH) and detection with anti-DNP antibodies. The degree of protein carbonylation was studied, also by derivatization and detection with anti-DNP, in primary cells of erythroblasts expanded for 11 days, by one-dimensional and two-dimensional electrophoresis (n = 16; 7 MDS, 6 controls, 2 reactive controls and 1 MDS + DFX). Expression levels of p21 were analyzed by qRT-PCR (n=19; 8C, 8 MDS, 3DFX).
RESULTS: Immunohistochemical assays revealed a higher level of carbonylation in patients with MDS (Fig. 1a). Interestingly, Deferasirox-treated MDS patients had lower levels than non-treated patients (Fig. 1b). In addition, to evaluate the carbonylation in the erythroid precursors, the pattern of carbonylated proteins in the expanded erythroblasts was analyzed. Again, a higher level of MDS was observed compared to the control group, which was reversed after treatment with Deferasirox. Two-dimensional electrophoresis revealed that differences in carbonylation are due to a limited number of proteins or aggregates, rather than to a large spectrum of proteins. Finally, an increased p21 expression was observed in patients with myelodysplasia and, surprisingly, this effect was reversed with Deferasirox treatment.
CONCLUSION: Patients with MDS showed a differential protein pattern respect to control patients, both in the myeloid and erythroid series. The increase in oxidative stress in myelodysplastic patients seems to activate signaling pathways involving p21. Treatment with Deferasirox can reverse the level of oxidation and the increase of p21 in patients with MDS.
Martinez Lopez:Celgene: Research Funding, Speakers Bureau; Jansen: Research Funding, Speakers Bureau; BMS: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau.
