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 μg 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.
| Original language | English |
|---|---|
| Pages (from-to) | 105-113 |
| Number of pages | 9 |
| Journal | Hematology Journal |
| Volume | 3 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - 2002 |
Fingerprint
Keywords
- Cord blood
- ERK dual-phosphorylation
- ERK sustained activity
- MEK dual-phosphorylation
- Proliferation of hematopoietic progenitors
- Self-renewing of hematopoietic progenitors
ASJC Scopus subject areas
- Hematology
Cite this
MEK-ERK pathway is expressed but not activated in high proliferating, self-renewing cord blood hematopoietic progenitors. / Bonati, Antonio; Lunghi, Paolo; Gammaitoni, Loretta; Pinelli, Silvana; Ridolo, Erminia; Dall'Aglio, Pier Paolo; Piacibello, Wanda; Aglietta, Massimo.
In: Hematology Journal, Vol. 3, No. 2, 2002, p. 105-113.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - MEK-ERK pathway is expressed but not activated in high proliferating, self-renewing cord blood hematopoietic progenitors
AU - Bonati, Antonio
AU - Lunghi, Paolo
AU - Gammaitoni, Loretta
AU - Pinelli, Silvana
AU - Ridolo, Erminia
AU - Dall'Aglio, Pier Paolo
AU - Piacibello, Wanda
AU - Aglietta, Massimo
PY - 2002
Y1 - 2002
N2 - 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 μg 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.
AB - 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 μg 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.
KW - Cord blood
KW - ERK dual-phosphorylation
KW - ERK sustained activity
KW - MEK dual-phosphorylation
KW - Proliferation of hematopoietic progenitors
KW - Self-renewing of hematopoietic progenitors
UR - http://www.scopus.com/inward/record.url?scp=0036001066&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0036001066&partnerID=8YFLogxK
U2 - 10.1038/sj/thj/6200162
DO - 10.1038/sj/thj/6200162
M3 - Article
C2 - 12032872
VL - 3
SP - 105
EP - 113
JO - Hematology Journal
JF - Hematology Journal
SN - 1466-4860
IS - 2
ER -