Granulocyte-macrophage colony-stimulating factor and interleukin-3 induce cell cycle progression through the synthesis of c-Myc protein by internal ribosome entry site-mediated translation via phosphatidylinositol 3-kinase pathway in human factor-dependent leukemic cells

Authors:
Kobayashi N, Saeki K and Yuo A
In:
Source: Blood
Publication Date: (2003)
Issue: 102(9): 3186-3195
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Cells used in publication:
UT7
Species: human
Tissue Origin: blood
F36P
Species: human
Tissue Origin: blood
M-07e
Species: human
Tissue Origin: blood
Platform:
Nucleofectorâ„¢ I/II/2b
Experiment

Cells of the factor-dependent human leukemic cell lines MO7e and F36P, arrested in G1 phase of the cell cycle, were nucleofected with different cistronic reporter gene constructs, each containing two distinguishable luciferase genes: Constructs that incorporated a c-myc gene sequence between the two luciferase genes, led to a 150 to 200 fold relative increase of the following luciferase gene, proving the strong activity of c-myc as an IRES in both cell lines. Constructs starting with an additional short palindromic sequence capable of building a hairpin inhibited translation of the first luciferase. The c-myc gene still increased the translation of the consecutive luciferase, ruling out the possibility of cap-dependent translation. Effects of hematopoietic growth factors were tested in both of the nucleofected cell lines. beta-galactosidase expression was used for normalization of the luciferase activity.

Abstract

Granulocyte-macrophage colony-stimulating factor and interleukin-3 induce cell cycle progression through the synthesis of c-Myc protein by internal ribosome entry site-mediated translation via phosphatidylinositol 3-kinase pathway in human factor-dependent leukemic cells. Kobayashi N, Saeki K, Yuo A. Department of Hematology, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan. To investigate the roles of c-myc during hematopoietic proliferation induced by growth factors, we used factor-dependent human leukemic cell lines (MO7e and F36P) in which proliferation, cell cycle progression, and c-Myc expression were strictly regulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3). In these cell lines, both c-myc mRNA and c-Myc protein stability were not affected by GM-CSF and IL-3, suggesting a regulation of c-Myc protein at the translational level. However, rapamycin, an inhibitor of cap-dependent translation, did not block c-myc induction by GM-CSF and IL-3. Thus, we studied the cap-independent translation, the internal ribosome entry site (IRES), during c-Myc protein synthesis using dicistronic reporter gene plasmids and found that GM-CSF and IL-3 activated c-myc IRES to initiate translation. c-myc IRES activation, c-Myc protein expression, and cell cycle progression were all blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002. In another factor-dependent cell line, UT7, we observed the cell cycle progression and up-regulation of c-Myc protein, c-myc mRNA, and c-myc IRES simultaneously, which were all inhibited by LY294002. Results indicate that hematopoietic growth factors induce cell cycle progression via IRES-mediated translation of c-myc though the PI3K pathway in human factor-dependent leukemic cells.