Prevention of apoptosis in J2E erythroid cells by erythropoietin: involvement of JAK2 but not MAP kinases

Abstract

The J2E erythroid cell line, transformed by retroviral v-raf/vmyc oncogenes, proliferates and differentiates in response to erythropoietin. Here we show that J2E cells undergo apoptosis rapidly after serum withdrawal and that only erythropoietin of seven growth factors tested, could protect the cells from death. The role of JAK2 and MAP kinases in transmitting viability signals initiated by erythropoietin was examined in these cells. Despite constituitive raf kinase activity, phosphorylation of MAP kinases fell after serum withdrawal. However, an antisense oligonucleotide strategy revealed that JAK2, but not the MAP kinases, was involved in transmitting signals to maintain the viability of J2E cells. Several cell cycle proteins and transcription factors were also studied; although c-jun rose sharply during apoptosis, erythropoietin could not suppress this increase. It was concluded that erythropoietin-induced protection from apoptosis involved JAK2, but not MAP kinases or c-jun.

Transcription Factor Erythroid Krüppel-like Factor (ELKF) Is Essential for the Erythropoietin-induced Hemoglobin Production but Not for Proliferation, Viability, or Morphological Maturation

“…Protein Analysis-Total cellular protein (50 g) was fractionated on 10 -12% polyacrylamide gels and transferred to nitrocellulose as we have described elsewhere (30,35,52). Immobilized proteins were immunoblotted with antibodies to EKLF (1, 4), GATA-1 (SC265, Santa Cruz Biotechnology, CA), BKLF (4), NF-E2 (45), globins (Cappel, Organon Tecknika, Belgium), phosphotyrosine (O5-321, Upstate Biotechnology, Lake Placid, NY), v-raf (SC133, Santa Cruz Biotechnology, CA, USA), and MAP-kinase (SC154, Santa Cruz Biotechnology).…”

“…Therefore, we have used the v-raf/v-myc-transformed J2E erythroleukemic cell line (27) as a model to investigate the role of EKLF in epo-stimulated differentiation. In response to epo, J2E cells undergo typical erythroid maturation characterized by activation of erythroid-specific genes, accumulation of functional hemoglobin, enhanced proliferation, maintenance of viability and morphological alteration (27)(28)(29)(30)(31). Thus, the importance of EKLF to each facet of erythroid differentiation could be assessed in this system.…”

Type I interferon differential therapy for erythroleukemia: specificity of STAT activation

“…51,53 Circulating expression levels of IFN transgenes in vivo have previously been found to be similar, with an average of 20 IU/mL sera. 49 …”

“…49 TUNEL assays were performed according to the manufacturer's instructions, staining with anti-BrdU antibody followed by streptavidin horseradish peroxidase and diaminobenzidine (DAB) substrate development. Cells were counterstained with hematoxylin, mounted, and viewed with light microscopy.…”

Adding self-renewal in committed erythroid progenitors improves the biological relevance of a mathematical model of erythropoiesis

“…The shape of the apoptosis rate function can be deduced from Chappell et al (1997), where the viability dose response of cells to EPO is presented, and Sakata et al (1985), where the relation between EPO concentration and mature erythrocytes density (via hemoglobin concentration) is described. This led to the Michaelis-Menten function given above to describe the implicit dependence of apoptosis on mature erythrocytes.…”