Overexpression of c-Myc or E2F1 sensitizes host cells to various types of apoptosis. Here, we found that overexpressed c-Myc or E2F1 induces accumulation of reactive oxygen species (ROS) and thereby enhances serum-deprived apoptosis in NIH3T3 and Saos-2. During serum deprivation, MnSOD mRNA was induced by NF-kappaB in mock-transfected NIH3T3, while this induction was inhibited in NIH3T3 overexpressing c-Myc or E2F1. In these clones, E2F1 inhibited NF-kappaB activity by binding to its subunit p65 in competition with a heterodimeric partner p50. In addition to overexpressed E2F1, endogenous E2F1 released from Rb was also found to inhibit NF-kappaB activity in a cell cycle-dependent manner by using E2F1(+/+) and E2F1(-/-) murine embryonic fibroblasts. These results indicate that E2F1 promotes apoptosis by inhibiting NF-kappaB activity.

Introduction c-Kit is a receptor tyrosine kinase (RTK), which constitutes a type III RTK subfamily with the receptors for platelet-derived growth factor (PDGF), colony-stimulating factor 1 (CSF-1), and flt-3 ligand. 1,2 The type III RTKs are characterized by an extracellular domain with 5 immunoglobulinlike domains and a cytoplasmic domain consisting of a kinase domain that is interrupted by a kinase insert. c-Kit (KIT) and its ligand stem cell factor (SCF) play an important role in hematopoiesis, melanogenesis, and gametogenesis, 3 as has been clearly shown by loss of function mutations of c-kit gene. In addition, c-kit gene product has been associated with various forms of neoplasms. Activating mutants of KIT, either in the juxtamembrane domain or the catalytic domain, were identified as the cause for transformation of hematopoietic stem cells, mast cells, and gastrointestinal stromal cells. [4][5][6][7][8][9][10] Thus, KIT/SCF has pleiotropic functions such as proliferation, survival, differentiation, and transformation. In this report, we focus on SCF/KITmediated cell migration, which is also a characteristic function of SCF in hematopoietic stem cells and mast cells, [11][12][13] and has critical roles in immunity, metastasis, and development.On ligand stimulation, KIT receptors dimerize, activate its intrinsic tyrosine kinase, and autophosphorylate. The phosphorylated KIT receptor generates binding sites for SH2 domaincontaining proteins, which include proteins of the p21Ras-mitogenactivated protein kinase (MAPK) pathway, 14 the p85 subunit of phosphatidylinositol 3Ј kinase (PI3K), 15 phospholipase C-␥ 1 , the Grb2 adaptor protein, 16 the Src family kinases (SFKs), 17 Cbl, CRKL, 19 SHP1, and SHP2. 20 Those proteins are subsequently activated or phosphorylated and further transduce signaling cascades that lead to various cellular responses. However, little is known about which signaling is essential for SCF-mediated migration. Recently, a few reports indicated that Lyn or p38 MAPK plays an important role, 21,22 but no comprehensive investigation has been done in which the tyrosine residue of KIT is involved in signal transduction, which is required for cell migration. In this study, we have converted all possible tyrosine (Y) residues on KIT cytoplasmic domain to phenylalanine (F) and introduced these YF substitute mutants on 293T cells or murine interleukin 3 (IL-3)-dependent BAF3 cells. We used these cell lines to elucidate signaling cascades that are important for SCF-mediated cell Supported in part by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology, the Japanese Ministry of Health, Labor and Welfare, and the Japan Society for Promotion of Science.Reprints: Yuzuru Kanakura, Department of Hematology and Oncology, Osaka University Graduate School of Medicine, 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan; e-mail: kanakura@bldon.med.osaka-u.ac.jp.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate thi...

Somatic mutation of PIGA in hematopoietic stem cells causes deficiency of glycosyl phosphatidylinositol-anchored proteins in paroxysmal nocturnal hemoglobinuria (PNH) that underlies the intravascular hemolysis but does not account for expansion of the PNH clone. Immune mechanisms may mediate clonal selection but appear insufficient to account for the clonal dominance necessary for PNH to become clinically apparent. Herein, we report 2 patients with PNH whose PIGAmutant cells had a concurrent, acquired rearrangement of chromosome 12. In both cases, der(12) had a break within the 3 untranslated region of HMGA2, the architectural transcription factor gene deregulated in many benign mesenchymal tumors, that caused ectopic expression of HMGA2 in the bone marrow. These observations suggest that aberrant HMGA2 expression, in concert with mutant PIGA, accounts for clonal hematopoiesis in these 2 patients and suggest the concept of PNH as a benign tumor of the bone marrow. ( IntroductionParoxysmal nocturnal hemoglobinuria (PNH) is a consequence of nonmalignant clonal expansion of hematopoietic stem cells with somatic mutation of PIGA. 1 Mutant PIGA 2 explains the deficiency of glycosyl phosphatidylinositol-anchored proteins (GPI-APs) that underlies the intravascular hemolysis of PNH. 3 However, PIGAmutant stem cells have no intrinsic proliferative advantage, 4,5 suggesting a 2-step model of pathogenesis.Step 1 of this model, clonal selection, 6,7 is envisioned as a conditional survival advantage that depends on deficiency of 1 or more GPI-APs. The close association of PNH with aplastic anemia, suggests that the selection pressure is immune mediated. 6,7 But, although 60% to 70% of patients with aplastic anemia have small, subclinical populations of GPI-AP Ϫ hematopoietic cells at diagnosis, 8 only 10% to 15% subsequently develop clinically apparent PNH. 9 In the remainder, GPI-AP Ϫ cells persist subclinically or disappear, 8 suggesting that mutant PIGA (and the consequent deficiency of GPI-APs) is necessary for clonal selection but is insufficient to account for the clonal expansion required for clinical manifestations of PNH to become apparent.Clonal expansion, step 2 of the PNH pathogenesis model, is envisioned as a consequence of clonal evolution in which a second somatic mutation bestows on the PIGA-mutant stem cell a proliferative advantage. 10 Herein, we present evidence supporting this 2-step model by showing a concurrent, acquired genetic abnormality in the PIGAmutant cells of 2 patients that establishes a novel mechanism for the nonmalignant clonal hematopoieis characteristic of PNH. Patients, materials, and methods PatientsInformed consent was obtained from patients J20 and US1 according to protocols approved by the Institutional Review Boards of Osaka University Hospital (Osaka, Japan) and the University of Utah School of Medicine (Salt Lake City, UT), respectively. Hybrid cell linesMonocytes derived from J20 or US1 were fused with the hypoxanthine phosphoribosyltransferase-negative mouse myeloma cell line, P...