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

Abstract

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.

Neuregulin 1-mediated ErbB2/ErbB3 signalling protects human trophoblasts against apoptosis to preserve differentiation

“…Downstream targets of the NRG1–ErbB2–ErbB3 axis include Akt1, mTOR, S6K and ERK1/2, all of which are related to survival in various cell types and model systems ( Kennedy et al, 1997 ; Xia et al, 1995 ). Interestingly, a study using constitutively active viral (v)-ErbB demonstrates that the PI3K–Akt and MAPK/ERK pathways are equally important in ErbB-mediated survival ( McCubrey et al, 2004 ). It has been further shown that only those cells, which rely on PI3K and MEK protein activity for proliferation, undergo apoptosis in the presence of PI3K and MEK inhibitors.…”

Isolation and characterization of dominant and recessive IL-3-independent hematopoietic transformants

“…IL-3 binding leads to b-chain autophosphorylation on tyrosine residues and activation of the Jak-Stat, mitogen-activated protein kinases (MAPK) and PI3K-/PKB pathways resulting in proliferation and inhibition of apoptosis (for references see Blalock et al (1999)). IL-3-independent growth has been observed via activating mutations of the common IL-3R b-subunit (D'Andrea et al, 1994;Hannemann et al, 1995;McCormack and Gonda, 1997) and a variety of mechanisms involving bcr-abl (Jiang et al, 2002), v-erbB (Shounan et al, 1995;McCubrey et al, 2004), v-src (Overell et al, 1987), c-kit (Piao and Bernstein, 1996), activated Stat5 (Onishi et al, 1998) pim-1 (Nosaka and Kitamura, 2002), Flt3 (Hayakawa et al, 2000), mpl (Onishi et al, 1996b) and H-ras (Andrejauskas and Moroni, 1989). For a recent review see Steelman et al (2004).…”

Viral mutations enhance the Max binding properties of the vMyc b-HLH-LZ domain

“…Indeed, comparative studies between these virally derived oncogenes and their cellular counterparts have contributed greatly toward our current understanding of their molecular mechanism of action. vSrc, the transforming component of Rous sarcoma virus, and vErbB, isolated from the avian erythroblastosis virus, AEV, contain several that contribute to their constitutive tyrosine kinase activity (1,2). …”