Proteomic Analysis of the Role of STAT5A in the Apoptosis of Human Pre-B Cells : Evidence for a Connection to Daxx and the Oxidative Stress

Abstract

It is well established that STAT5A and 5B transcription factors (TF) play a major role in hematopoiseis and oncogenesis. In particular, there is a body of evidence suggesting that they might be involved in B lymphocyte survival, proliferation and development as well as in B cell neoplastic transformation. To investigate the role of STAT5A in human precursor B cell survival, we stably transfected a dominant negative form of STAT5A (DN-STAT5A, deleted in its transactivation domain) in the human leukemic Nalm6 pre-B cell line. All clones expressing DN-STAT5A exhibited a lower proliferation rate associated with a higher spontaneous apoptosis and that was massively enhanced upon IL7 stimulation. They also were more sensitive to FAS (CD95)- and at a lesser extent to etoposide-induced cell death than cells transfected with the empty vector (Nalm6neo), suggesting a hitherto unknown link between STAT5A and apoptosis/survival pathways. There was no evidence for changes in the levels of expression of Bcl2, BclxL, Bad or –c-Myc in DN-STAT5A expressing cells whereas Bax and Bim expression was sharply up-regulated in these cells. In order to identify the proteins involved in the connection between Fas and STAT5A, we used a proteomic approach with differential analysis of cells expressing (Nalm6Δ5A749) or not (Nalm6neo) the DN-STAT5A. Statistical analysis of bidimensional (2D) gels and use of MALDI-TOF technique enabled us to identify 7 proteins down-regulated in the Nalm6Δ5A749 cells, including members of heat shock proteins such as hsp27 and hsp70, as well as proteins implied in the control of oxidative stress like glutathione synthetase and transaldolase. By contrast, whereas 6 proteins were shown to be up-regulated, including prohibitin which is a negative regulator of cellular proliferation. Nalm6Δ5A749 cells exhibited enhanced levels of reactive oxygen species in keeping with the idea that oxidative stress might be involved in the increased sensitivity of these cells to apoptotic signals. Although expression of all above mentioned molecules did not change upon Fas stimulation, addition of gluthatione (10mM) resulted in a complete inhibition of Fas-mediated apoptosis in Nalm6Δ5A749 cells. Whether or not prohibitin might be involved in the decreased proliferation rate in DN-STAT5A expressing cells needs further investigation. As Hsp27 has been previously shown to have the potential to sequestrate Daxx, a molecule that is involved in Fas receptor signaling, we investigated the subcellular location of this molecule in Nalm6Δ5A749 cells. We showed that Daxx was essentially expressed in the cytoplasm of these cells whereas it was mainly located in the nucleus in Nalm6neo cells. Our results bring evidence for a role of STAT5A in pre-B cell growth and survival and in particular for a connection between this TF and Fas signaling machinery that might involve Hsp27 and Daxx. They also point out to a so far undescribed link between STAT5 and oxidative stress.