INTRODUCTION: The expression, activity and functions of mitogen-activated protein (MAP) kinases in primary human hematopoietic progenitors (HP) have not yet been fully clarified. MATERIAL AND METHODS: To perform our experiments we used a stroma-free cell culture system in which the combination of FLT3 ligand (FL), stem cell factor (SCF) and thrombopoietin (TPO) induces massive expansion and proliferation of cord blood HP. The addition of IL-3 results in a rapid decrease of HP due to the prevalence of maturation and cell death. To detect extracellular regulated kinase (ERK) immunoenzymatic activity we recovered HP from FL, SCF and TPO stimulated long term cultures (LTC) after four weeks of culture. Some samples were recovered 16 h after addition of IL-3 to the LTC. We selectively immunoprecipitated p44/42 ERK kinase from 245 microg of cell lysates. We then analysed dual-phosphorylation of ERK-activating kinase-kinase (p45 MEK1/2) and of p44 ERK1 and p42 ERK2, and investigated MEK and ERK expression. RESULTS: ERK activity, MEK1, and p42 and p44 ERK dual-phosphorylation were undetectable in the expanding, greatly proliferating and self-renewing HP. However, after addition of IL-3 sustained (still detectable 16 h after the stimulus) and high levels of ERK activity and dual-phosphorylation of the kinases were seen. The levels of MEK and ERK expression were stable in the different phases. CONCLUSIONS: These findings add new information on the intracellular mechanisms of HP and help explain the very low levels of hematopoietic toxicity recently seen when treating cancer with down-modulators of ERK activity.
MEK-ERK pathway is expressed but not activated in high proliferating, self-renewing cord blood hematopoietic progenitors.
PIACIBELLO, Vanda;AGLIETTA, Massimo
2002-01-01
Abstract
INTRODUCTION: The expression, activity and functions of mitogen-activated protein (MAP) kinases in primary human hematopoietic progenitors (HP) have not yet been fully clarified. MATERIAL AND METHODS: To perform our experiments we used a stroma-free cell culture system in which the combination of FLT3 ligand (FL), stem cell factor (SCF) and thrombopoietin (TPO) induces massive expansion and proliferation of cord blood HP. The addition of IL-3 results in a rapid decrease of HP due to the prevalence of maturation and cell death. To detect extracellular regulated kinase (ERK) immunoenzymatic activity we recovered HP from FL, SCF and TPO stimulated long term cultures (LTC) after four weeks of culture. Some samples were recovered 16 h after addition of IL-3 to the LTC. We selectively immunoprecipitated p44/42 ERK kinase from 245 microg of cell lysates. We then analysed dual-phosphorylation of ERK-activating kinase-kinase (p45 MEK1/2) and of p44 ERK1 and p42 ERK2, and investigated MEK and ERK expression. RESULTS: ERK activity, MEK1, and p42 and p44 ERK dual-phosphorylation were undetectable in the expanding, greatly proliferating and self-renewing HP. However, after addition of IL-3 sustained (still detectable 16 h after the stimulus) and high levels of ERK activity and dual-phosphorylation of the kinases were seen. The levels of MEK and ERK expression were stable in the different phases. CONCLUSIONS: These findings add new information on the intracellular mechanisms of HP and help explain the very low levels of hematopoietic toxicity recently seen when treating cancer with down-modulators of ERK activity.
I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
