Abrogation of cytokine-dependency by MEK1 involves the PI3 kinase-AKT pathway and is enhanced by BCL2 overexpression or aberrant AKT activation

Research output: Contribution to journalArticle

Abstract

The Ra.s/Raf/MEK/MAPK pathway is activated in hematopoietic cells in response to cytokines such as IL-3 and GM-CSF. This pathway is intimately associated with cell growth and the regulation of apoptosis. Since Ras mutations are present in 10-50% of myelodysplastic disease and acute myelogenous leukemias and altered Ras activity ultimately results in the subsequent activation of Raf, MEK and MAPK, we sought to examine what role(s) activation of the serine/threonine and tyrosine kinase MEK1 had on hematopoietic cell growth and regulation of apoptosis. Using a retrovirus encoding AMEKlrER, an estradiol-inducible form of MEK1, we investigated the ability of this protein to abrogate the cytokine-dependency of murine FDC-P1 cells. Cytokineindependent FDC-P1 cells were isolated at a frequency of 1 in 20,000 AMEKliERinfected cells and maintained in β-estradiol. Removal of hormone elicited cessation of cell growth and apoptotic cell death. Furthermore, treatment with the MEK1 inhibitor, PD98059, or the PI3 kinase inhibitor, LY294002, suppressed AMEKhER-mediated proliferation. Activation of AMEK1:ER led to rapid activation of the ERK and PI3 kinases as well as the anti-apoptotic kinase AKT. Overexpression of BCL2 in cytokinedependent AMEKl:ER-infected cells enhanced the recovery of MEK1-responsive cells by 10-fold. Moreover, Overexpression of BCL2 resulted in prolonged MEK1 and ERK activity, as well as a higher percentage of cells in S phase after the removal of β-estradiol. In addition, expression of a myristolated, conditionally-active form of AKT, AKT:ER, enhanced the recovery of cytokine-independent cells 100-fold in synergy with AMEK1 :ER. A decreased level of apoptosis and enhanced drug-resistance was observed in MEK1responsive cells overexpressing BCL2 or AKT:ER, as determined by DNA fragmentation and Annexin V/propidium iodide staining. Expression of BCL2 or AKT:ER alone was unable to relieve cytokine-dependency of the FDC-P1 cells. AMEK1 :ER activation induced autocrine GM-CSF expression in the MEKl-responsive cells but not in the cytokinedependent cells. These data indicate that AMEK1:ER can abrogate the cytokinedependency of FDC-P1 cells with low efficiency. This abrogation of cytokine-dependency involves the activation of AKT and can be enhanced by Overexpression of BCL2 or the aberrant activation of AKT. These cell lines will be useful for elaborating the ability of MEK1 to regulate both the cell cycle and apoptotic machinery in hematopoietic cells.

Original languageEnglish
JournalBlood
Volume96
Issue number11 PART I
Publication statusPublished - 2000

Fingerprint

Phosphatidylinositol 3-Kinases
Chemical activation
Cytokines
Cell growth
Estradiol
Mitogen-Activated Protein Kinase Kinases
Granulocyte-Macrophage Colony-Stimulating Factor
Apoptosis
Cells
Recovery
Propidium
Interleukin-3
Annexin A5
Protein-Serine-Threonine Kinases
Cell death
Protein-Tyrosine Kinases
Machinery
Phosphotransferases
Hormones
Growth

ASJC Scopus subject areas

  • Hematology

Cite this

@article{a675b9b8af304675a988c307c2e23dd6,
title = "Abrogation of cytokine-dependency by MEK1 involves the PI3 kinase-AKT pathway and is enhanced by BCL2 overexpression or aberrant AKT activation",
abstract = "The Ra.s/Raf/MEK/MAPK pathway is activated in hematopoietic cells in response to cytokines such as IL-3 and GM-CSF. This pathway is intimately associated with cell growth and the regulation of apoptosis. Since Ras mutations are present in 10-50{\%} of myelodysplastic disease and acute myelogenous leukemias and altered Ras activity ultimately results in the subsequent activation of Raf, MEK and MAPK, we sought to examine what role(s) activation of the serine/threonine and tyrosine kinase MEK1 had on hematopoietic cell growth and regulation of apoptosis. Using a retrovirus encoding AMEKlrER, an estradiol-inducible form of MEK1, we investigated the ability of this protein to abrogate the cytokine-dependency of murine FDC-P1 cells. Cytokineindependent FDC-P1 cells were isolated at a frequency of 1 in 20,000 AMEKliERinfected cells and maintained in β-estradiol. Removal of hormone elicited cessation of cell growth and apoptotic cell death. Furthermore, treatment with the MEK1 inhibitor, PD98059, or the PI3 kinase inhibitor, LY294002, suppressed AMEKhER-mediated proliferation. Activation of AMEK1:ER led to rapid activation of the ERK and PI3 kinases as well as the anti-apoptotic kinase AKT. Overexpression of BCL2 in cytokinedependent AMEKl:ER-infected cells enhanced the recovery of MEK1-responsive cells by 10-fold. Moreover, Overexpression of BCL2 resulted in prolonged MEK1 and ERK activity, as well as a higher percentage of cells in S phase after the removal of β-estradiol. In addition, expression of a myristolated, conditionally-active form of AKT, AKT:ER, enhanced the recovery of cytokine-independent cells 100-fold in synergy with AMEK1 :ER. A decreased level of apoptosis and enhanced drug-resistance was observed in MEK1responsive cells overexpressing BCL2 or AKT:ER, as determined by DNA fragmentation and Annexin V/propidium iodide staining. Expression of BCL2 or AKT:ER alone was unable to relieve cytokine-dependency of the FDC-P1 cells. AMEK1 :ER activation induced autocrine GM-CSF expression in the MEKl-responsive cells but not in the cytokinedependent cells. These data indicate that AMEK1:ER can abrogate the cytokinedependency of FDC-P1 cells with low efficiency. This abrogation of cytokine-dependency involves the activation of AKT and can be enhanced by Overexpression of BCL2 or the aberrant activation of AKT. These cell lines will be useful for elaborating the ability of MEK1 to regulate both the cell cycle and apoptotic machinery in hematopoietic cells.",
author = "Blalock, {William L.}",
year = "2000",
language = "English",
volume = "96",
journal = "Blood",
issn = "0006-4971",
publisher = "American Society of Hematology",
number = "11 PART I",

}

TY - JOUR

T1 - Abrogation of cytokine-dependency by MEK1 involves the PI3 kinase-AKT pathway and is enhanced by BCL2 overexpression or aberrant AKT activation

AU - Blalock, William L.

PY - 2000

Y1 - 2000

N2 - The Ra.s/Raf/MEK/MAPK pathway is activated in hematopoietic cells in response to cytokines such as IL-3 and GM-CSF. This pathway is intimately associated with cell growth and the regulation of apoptosis. Since Ras mutations are present in 10-50% of myelodysplastic disease and acute myelogenous leukemias and altered Ras activity ultimately results in the subsequent activation of Raf, MEK and MAPK, we sought to examine what role(s) activation of the serine/threonine and tyrosine kinase MEK1 had on hematopoietic cell growth and regulation of apoptosis. Using a retrovirus encoding AMEKlrER, an estradiol-inducible form of MEK1, we investigated the ability of this protein to abrogate the cytokine-dependency of murine FDC-P1 cells. Cytokineindependent FDC-P1 cells were isolated at a frequency of 1 in 20,000 AMEKliERinfected cells and maintained in β-estradiol. Removal of hormone elicited cessation of cell growth and apoptotic cell death. Furthermore, treatment with the MEK1 inhibitor, PD98059, or the PI3 kinase inhibitor, LY294002, suppressed AMEKhER-mediated proliferation. Activation of AMEK1:ER led to rapid activation of the ERK and PI3 kinases as well as the anti-apoptotic kinase AKT. Overexpression of BCL2 in cytokinedependent AMEKl:ER-infected cells enhanced the recovery of MEK1-responsive cells by 10-fold. Moreover, Overexpression of BCL2 resulted in prolonged MEK1 and ERK activity, as well as a higher percentage of cells in S phase after the removal of β-estradiol. In addition, expression of a myristolated, conditionally-active form of AKT, AKT:ER, enhanced the recovery of cytokine-independent cells 100-fold in synergy with AMEK1 :ER. A decreased level of apoptosis and enhanced drug-resistance was observed in MEK1responsive cells overexpressing BCL2 or AKT:ER, as determined by DNA fragmentation and Annexin V/propidium iodide staining. Expression of BCL2 or AKT:ER alone was unable to relieve cytokine-dependency of the FDC-P1 cells. AMEK1 :ER activation induced autocrine GM-CSF expression in the MEKl-responsive cells but not in the cytokinedependent cells. These data indicate that AMEK1:ER can abrogate the cytokinedependency of FDC-P1 cells with low efficiency. This abrogation of cytokine-dependency involves the activation of AKT and can be enhanced by Overexpression of BCL2 or the aberrant activation of AKT. These cell lines will be useful for elaborating the ability of MEK1 to regulate both the cell cycle and apoptotic machinery in hematopoietic cells.

AB - The Ra.s/Raf/MEK/MAPK pathway is activated in hematopoietic cells in response to cytokines such as IL-3 and GM-CSF. This pathway is intimately associated with cell growth and the regulation of apoptosis. Since Ras mutations are present in 10-50% of myelodysplastic disease and acute myelogenous leukemias and altered Ras activity ultimately results in the subsequent activation of Raf, MEK and MAPK, we sought to examine what role(s) activation of the serine/threonine and tyrosine kinase MEK1 had on hematopoietic cell growth and regulation of apoptosis. Using a retrovirus encoding AMEKlrER, an estradiol-inducible form of MEK1, we investigated the ability of this protein to abrogate the cytokine-dependency of murine FDC-P1 cells. Cytokineindependent FDC-P1 cells were isolated at a frequency of 1 in 20,000 AMEKliERinfected cells and maintained in β-estradiol. Removal of hormone elicited cessation of cell growth and apoptotic cell death. Furthermore, treatment with the MEK1 inhibitor, PD98059, or the PI3 kinase inhibitor, LY294002, suppressed AMEKhER-mediated proliferation. Activation of AMEK1:ER led to rapid activation of the ERK and PI3 kinases as well as the anti-apoptotic kinase AKT. Overexpression of BCL2 in cytokinedependent AMEKl:ER-infected cells enhanced the recovery of MEK1-responsive cells by 10-fold. Moreover, Overexpression of BCL2 resulted in prolonged MEK1 and ERK activity, as well as a higher percentage of cells in S phase after the removal of β-estradiol. In addition, expression of a myristolated, conditionally-active form of AKT, AKT:ER, enhanced the recovery of cytokine-independent cells 100-fold in synergy with AMEK1 :ER. A decreased level of apoptosis and enhanced drug-resistance was observed in MEK1responsive cells overexpressing BCL2 or AKT:ER, as determined by DNA fragmentation and Annexin V/propidium iodide staining. Expression of BCL2 or AKT:ER alone was unable to relieve cytokine-dependency of the FDC-P1 cells. AMEK1 :ER activation induced autocrine GM-CSF expression in the MEKl-responsive cells but not in the cytokinedependent cells. These data indicate that AMEK1:ER can abrogate the cytokinedependency of FDC-P1 cells with low efficiency. This abrogation of cytokine-dependency involves the activation of AKT and can be enhanced by Overexpression of BCL2 or the aberrant activation of AKT. These cell lines will be useful for elaborating the ability of MEK1 to regulate both the cell cycle and apoptotic machinery in hematopoietic cells.

UR - http://www.scopus.com/inward/record.url?scp=33748658472&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33748658472&partnerID=8YFLogxK

M3 - Article

VL - 96

JO - Blood

JF - Blood

SN - 0006-4971

IS - 11 PART I

ER -