Role of Dok-1 and Dok-2 in Myeloid Homeostasis and Suppression of Leukemia

Dok-1 and Dok-2 are closely related rasGAP-associated docking proteins expressed preferentially in hematopoietic cells. Although they are phosphorylated upon activation of many protein tyrosine kinases (PTKs), including those coupled with cytokine receptors and oncogenic PTKs like Bcr-Abl, their physiological roles are largely unidentified. Here, we generated mice lacking Dok-1 and/or Dok-2, which included the double-deficient mice succumbed to myeloproliferative disease resembling human chronic myelogenous leukemia (CML) and chronic myelomonocytic leukemia. The double-deficient mice displayed medullary and extramedullary hyperplasia of granulocyte/macrophage progenitors with leukemic potential, and their myeloid cells showed hyperproliferation and hypo-apoptosis upon treatment and deprivation of cytokines, respectively. Consistently, the mutant myeloid cells showed enhanced Erk and Akt activation upon cytokine stimulation. Moreover, loss of Dok-1 and/or Dok-2 induced blastic transformation of chronic phase CML-like disease in mice carrying the bcr-abl gene, a cause of CML. These findings demonstrate that Dok-1 and Dok-2 are key negative regulators of cytokine responses and are essential for myeloid homeostasis and suppression of leukemia

Prevention of Ornithine Cytotoxicity by Nonpolar Side Chain Amino Acids in Retinal Pigment Epithelial Cells

PURPOSE. To investigate the effect of amino acids on ornithine cytotoxicity in ornithine-␦-aminotransferase (OAT)-deficient human retinal pigment epithelial (RPE) cells as an in vitro model of gyrate atrophy (GA) of the choroid and retina. METHODS. RPE cells were treated with 0.5 mM 5-fluoromethylornithine (5-FMOrn), a specific and irreversible OAT inhibitor. OAT-deficient RPE cells were incubated with 10 mM ornithine in the presence of 20 mM of 1 of 18 amino acids or 10 mM 2-amino-2-norbornane-carboxylic acid (BCH), a conventional inhibitor of the amino acid transporter system L. Ornithine cytotoxicity and cytoprotective effects of each amino acid was evaluated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay 72 hours after treatment with ornithine in OAT-deficient RPE cells. Ornithine incorporation into RPE cells was evaluated using DL discovered that the biochemical abnormalities of this disorder are hyperornithinemia and overflow ornithinuria. Further, the finding of an association between hyperornithinemia and GA led to the discovery of an enzyme defect, a deficiency of the mitochondrial matrix enzyme ornithine-␦-aminotransferase (OAT). 3-5 We have reported that inactivation of OAT in human retinal pigment epithelial (RPE) cells by 5-fluoromethylornithine (5-FMOrn), a specific irreversible inhibitor of OAT, 6 makes them susceptible to ornithine, leading to cell death. 7 A human hepatoma cell line, HepG2, and a fibroblast cell line, WI-38, which possess OAT activity comparable to that of RPE cells, are not affected by ornithine when OAT is inactivated by 5-FMOrn, suggesting that ornithine cytotoxicity toward 5-FMOrn-treated RPE can be used as an in vitro model of GA. We have also demonstrated that proline prevents ornithine-induced cell death. Mammalian cells have two biosynthetic pathways for proline and share a common intermediate, L-⌬ 1 -pyrroline-5-carboxylic acid (P5C), which is formed from ornithine by OAT and from glutamic acid by P5C synthase. In a previous study, ornithine almost completely blocked P5C synthase and decreased the incorporation of proline derived from glutamic acid into protein in fibroblasts cultured from patients with GA 8 ; however, the mechanisms of ornithine cytotoxicity in OAT-deficient RPE cells and cytoprotection by proline remain to be elucidated. In the course of our earlier experiments, we noticed that 5-FMOrn-treated RPE cells are damaged by ornithine when cultured in Ham F12 medium, whereas they are not affected by ornithine in Dulbecco's modified Eagle's (DME) medium devoid of proline. Those unexpected results prompted us to investigate the effect of other amino acids on ornithine cytotoxicity and incorporation of ornithine in human RPE cells to clarify the mechanisms of ornithine cytotoxicity. From the present results, we report that most of the nonpolar and uncharged polar side chain amino acids tested prevented ornithine cytotoxicity through L-type amino acid transporters. MATERIAL AND METHODS Cell Culture A human RPE cell line, hTERT-RPE, previously established by gene transfer of human telomerase reverse transcriptase cDNA, 9 was kindly provided by Donald J. Zack (Wilmer Ophthalmological Institute, Johns Hopkins University, Baltimore, MD). This RPE cell line has been reported to have several characteristics of other normal RPE cells, such as an expression of Rpe65 and in vitro differentiation capacity. From the Departments of 1 Ophthalmology an