Wayne C. Glasgow

Cellular proliferation and lipid metabolism: importance of lipoxygenases in modulating epidermal growth factor-dependent mitogenesis

In this article we have reviewed and discussed the results of our investigation of lipid metabolites as modulators of epidermal growth factor (EGF) signaling pathways. We have studied epidermal growth factordependent mitogenesis in BALB/c 3T3 and Syrian hamster embryo (SHE) cells in culture. We observed that EGF stimulates the formation of prostaglandins in BALB/c 3T3 cells and their formation appears to be necessary for EGF dependent mitogenesis. EGF did not stimulate PGE2 formation in SHE cells and in fact, exogenously added PGE2 inhibited mitogenesis. In both cell lines, EGF stimulated the formation of lipoxygenasederived 13(S)-hydroxyoctadecadienoic acid (13-HODE) and inhibition of 13-HODE formation attenuated mitogenesis. The addition of 13-(S)-HODE enhanced EGF-dependent mitogenesis but when added alone, the compound was not mitogenic. Other metabolites, including lipoxygenase metabolites of arachidonic acid, were either weak simulators of EGF-dependent mitogenesis or essentially inactive. The 13(S)-HODE appears to be formed by an apparently unique lipoxygenase that is regulated by the tyrosine kinase activity of the EGF receptor. The mechanisms by which lipids, particularly the lipoxygenase-derived linoleic acid metabolites, modulate the EGF signaling pathways leading to cell proliferation is discussed. The possible significance of lipoxygenase and prostaglandin H synthase-dependent metabolism of unsaturated fatty acids in breast and colon cancer is also discussed.

Expression of Gab1 Lacking the Pleckstrin Homology Domain Is Associated with Neoplastic Progression

ABSTRACT An in vitro transformation system of carcinogen-treated Syrian hamster embryo (SHE) cell cultures represents multistep genetic and nongenetic changes that develop during the neoplastic progression of normal cells to tumor cells in vivo. During this neoplastic progression, SHE cells demonstrate an altered response to epidermal growth factor (EGF). In the present report, we examined the role of the adapter protein Gab1 (Grb2-associated binder-1) in the neoplastic progression of SHE cells. We used two asbestos-transformed SHE cell clones in different neoplastic stages: a 10W+8 clone, which is immortal and retains the ability to suppress the tumorigenicity of tumor cells in cell-cell hybrid experiments, and a 10W−1 clone, which has lost this tumor suppressor ability. 10W+8 cells expressed full-length 100-kDa Gab1 and associated 5.2-kb mRNA. Upon repeated cell passaging, 10W−1 cells showed increasing expression of a novel 87-kDa form of Gab1 as well as 4.6-kb mRNA with diminishing expression of the original 100-kDa Gab1. cDNA encoding the 87-kDa Gab1 predicts a form of Gab1 lacking the amino-terminal 103 amino acids (Gab1Δ1-103), which corresponds to loss of most of the pleckstrin homology (PH) domain. Gab1Δ1-103 retains the ability to be phosphorylated in an EGF-dependent manner and to associate with the EGF receptor and SHP-2 upon EGF stimulation. The endogenous expression of Gab1Δ1-103 in 10W−1 cells appeared closely related to EGF-dependent colony formation in soft agar. Moreover, transfection and expression of Gab1Δ1-103, but not Gab1, in 10W+8 cells enhanced their EGF-dependent colony formation in soft agar. These results demonstrate that Gab1 is a target of carcinogen-induced transformation of SHE cells and that the expression of a Gab1 variant lacking most of the PH domain plays a specific role in the neoplastic progression of SHE cells.

Modulation of Epidermal Growth Factor Signal Transduction by Linoleic Acid Metabolites

This laboratory is studying arachidonic acid and linoleic acid metabolites formed by prostaglandin H synthase and lipoxygenases as modulators of growth factor signaling pathways that lead to cellular proliferation. Growth factors bind to specific receptors located on the cell surface and initiate a series of biochemical events that ultimately leads to cellular proliferation. Occupation of the receptor by growth factor causes dimerization of the receptor and stimulates the receptor’s intrinsic tyrosine kinase activity. The kinase activity phosphorylates key tyrosine residues present in several intracellular signaling proteins and initiates a series of tyrosine, serine and threonine phosphorylation of proteins that link the cell surface with the nucleus. Phosphorylation of these signaling proteins regulates their catalytic activity and the association to other signaling proteins. The final step in the signaling cascade appears to be activation of mitogen-activated protein kinase (MAP kinase) which in turn activates transcription factors. The nature of this intracellular communication is poorly understood and thus is the subject of intense study. Several lines of evidence suggest that prostaglandin H synthase and lipoxygenase catalyzed metabolites of arachidonic acid and linoleic acid modulate the epidermal growth factor (EGF) mitogenic signal in fibroblasts.

The Role of Linoleic Acid Metabolism in the Proliferative Response of Cells Overexpressing the erbB-2/HER2 Oncogene

The polypeptide mitogen epidermal growth factor (EGF) stimulates quiescent cells to initiate DNA synthesis and cell division. Binding of EGF to specific cell surface receptors activates a variety of biochemical pathways. The role of each of these biochemical processes in transducing the mitogenic signal has yet to be clearly delineated. Arachidonic and linoleic acid metabolism have been implicated in modulating cell growth or cell transformation. Increased production of these lipid compounds is associated with many tumor cell types and these products are described as regulating immune function, tumor growth, promotion, and metastasis.

Structural Requirements for Enhancement of EGF-Dependent DNA Synthesis by Oxygenated Metabolltes of Linoleic Acid

Epidemiological and animal model studies suggest a link between high dietary fat intake and the etiology of colon and breast cancer (1,2). In particular, linoleic acid, as the major polyunsaturated fatty acid consumed by humans, enhances tumorigenesis in several model systems (3–5). Our previous work suggests a role for specific oxygenated metabolites of linoleic acid in modulating cell proliferation and/or transformation (6,7). In Syrian hamster embryo (SHE) fibroblasts, we observed potentiation of epidermal growth factor (EGF)-dependent mitogenesis by 13-hydroxyoctadecadienoic acid (HODE), the primary linoleate metabolite formed in these cells. The mechanism of action by which linoleate derivatives enhance EGF-regulated [3H]thymidine incorporation has not been established. As an initial approach to investigate this area, we examined the structural characteristics of bioactive lipid compounds related to stimulation of DNA synthesis in SHE cells.

Role of Archidonic Acid and Linoleic Acid Metabolism in Epidermal Growth Factor Initiated Proliferation of Syrian Hamster Embryo Fibroblasts

Epidermal growth factor (EGF) binds to specific receptors located on the cell surface and initiates a series of biochemical events that ultimately leads to cellular proliferation. Occupation of EGF receptor by EGF stimulates the receptor’s intrinsic tyrosine kinase activity which phosphorylates key tyrosine residues present in several intracellular signaling proteins that link the cell surface with the nucleus. Phosphorylation of these signaling proteins regulates their catalytic activity. The nature of this intracellular communication is poorly understood and thus is the subject of intense study. Several lines of evidence suggest the prostaglandin H synthase and lipoxygenase catalyzed metabolites of arachidonic acid and linoleic acid modulate the EGF mitogenic signal in fibroblasts.