Effects of a conditionally active v-ErbB and an EGF-R inhibitor on transformation of NIH-3T3 cells and abrogation of cytokine dependency of hematopoietic cells.
Epidermal growth factor (EGF) and its cognate receptor (EGF-R) are often dysregulated in human neoplasia. Moreover, EGF-R-transformed cell lines have constitutive EGF-R activity, which makes elucidation of its effects difficult to determine. In the following studies, the effects of a novel conditionally activated form of EGF-R, v-ErbB:ER, on the morphological transformation of NIH-3T3 cells and the abrogation of hematopoietic cell cytokine dependence were investigated. The v-ErbB ES-4 oncogene was fused to the hormone binding domain of the estrogen receptor (ER). This construct, v-ErbB:ER, requires beta-estradiol or 4-OH tamoxifen for activation. v-ErbB:ER conditionally transformed NIH-3T3 cells and abrogated cytokine dependence of hematopoietic cells. Stimulation of v-ErbB:ER activity resulted in the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and Raf/MEK/ERK kinase cascades. To determine the importance of these signal transduction pathways, the conditionally transformed hematopoietic cells were treated with EGF-R, PI3K and MEK inhibitors. The EGF-R inhibitor AG1478 effectively inhibited MEK, ERK and Akt activation, and induced apoptosis when the cells were grown in response to v-ErbB:ER. Apoptosis was observed at 100- to 1000-fold lower concentrations of AG1478 when the cells were grown in response to v-ErbB:ER as opposed to IL-3. Furthermore, the parental, BCR-ABL- and Raf-transformed cells were only susceptible to the apoptosis-inducing effects of AG1478 at the highest concentrations demonstrating the specificity of these inhibitors. MEK or PI3K inhibitors suppressed ERK or Akt activation, respectively, and induced apoptosis in the v-ErbB:ER-responsive cells. However, MEK and PI3K inhibitors only induced apoptosis at 1000-fold higher concentrations than the EGFR inhibitor. This novel v-ErbB:ER construct and these conditionally transformed cell lines will be useful to further elucidate ErbB-mediated signal transduction and to determine the effectiveness of various inhibitors in targeting different aspects of EGF-R-mediated signal transduction and malignant transformation.
McCubrey
James A
JA
Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University, Greenville, NC 27858, USA. mccubreyj@mail.ecu.edu
Shelton
John G
JG
Steelman
Linda S
LS
Franklin
Richard A
RA
Sreevalsan
Thazepadath
T
McMahon
Martin
M
D016475:3T3 Cells; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001665:Binding Sites; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D016207:Cytokines / Q000502:physiology*; D004261:DNA Replication / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D018773:Genes, erbB-1; D006412:Hematopoietic Stem Cells / Q000276:immunology / Q000502:physiology*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D051379:Mice; D011799:Quinazolines; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology
D016207:Cytokines; D004791:Enzyme Inhibitors; D066246:ErbB Receptors; D011799:Quinazolines; D018719:Erb-b2 Receptor Tyrosine Kinases; D020032:Tyrphostins; C101044:RTKI cpd