ICSBP/IRF-8 inhibits mitogenic activity of p210 Bcr/Abl in differentiating myeloid progenitor cells by Tomohiko Tamura, Hee Jeong Kong, Chainarong Tunyaplin, Hideki Tsujimura, Kathryn Calame, and Keiko Ozato Blood Volume 102(13):4547-4554 December 15, 2003 ©2003 by American Society of Hematology

Transformation of ICSBP-/- myeloid progenitor cells by p210 Bcr/Abl. Transformation of ICSBP-/- myeloid progenitor cells by p210 Bcr/Abl. (A) Wright-Giemsa stain of parental Tot2 cells and Tot2 cells transduced with p210 Bcr/Abl retrovirus (Tot2p210 cells) (original magnification × 600). Tot2 cells were cultured in media containing GM-CSF, whereas Tot2p210 cells were cultured without GM-CSF. (B) Cell cycle analysis. Tot2 and Tot2p210 cells were analyzed for DNA content. (C) Immunoblot analysis for p210 Bcr/Abl and phosphotyrosine-containing proteins. (D) Effect of a Bcr/Abl kinase inhibitor, imatinib mesylate (STI571), on the growth and survival of Tot2 and Tot2p210 cells. Imatinib mesylate was added at indicated concentrations and viable cell number was monitored. Tomohiko Tamura et al. Blood 2003;102:4547-4554 ©2003 by American Society of Hematology

ICSBP induction of macrophage differentiation in Bcr/Abl-transformed ICSBP-/- myeloid progenitor cells. ICSBP induction of macrophage differentiation in Bcr/Abl-transformed ICSBP-/- myeloid progenitor cells. (A) Wright-Giemsa stain of Tot2 and Tot2p210 cells transduced with the control or ICSBP retrovirus on day 6 after transduction (original magnification × 600). (B) Semiquantitative reverse transcriptase-PCR (RT-PCR) analysis for macrophage differentiation-related genes. Expression of scavenger receptor (SR) and myeloperoxidase (MPO) transcripts was analyzed using RNA samples from indicated days after transduction. β-Actin was used as a control for equal loading. (C) Induction of LPS/IFNγ-responsive genes. Cells on 6 days after transduction were treated with 200 ng/mL LPS or 200 U/mL IFNγ for 6 hours. Indicated transcripts were analyzed by semiquantitative RT-PCR. iNOS indicates inducible nitric oxide synthase; and FcγRI, Fcγ receptor I. Tomohiko Tamura et al. Blood 2003;102:4547-4554 ©2003 by American Society of Hematology

Cell growth arrest by ICSBP in Bcr/Abl-transformed ICSBP-/- myeloid progenitor cells. Cell growth arrest by ICSBP in Bcr/Abl-transformed ICSBP-/- myeloid progenitor cells. (A) Growth rate of Tot2 and Tot2p210 cells transduced with the indicated viruses. Total cell yields are counted on the indicated days. (B) Cell cycle analysis. Cells transduced with control or ICSBP retrovirus were analyzed for DNA contents on the indicated days. Tomohiko Tamura et al. Blood 2003;102:4547-4554 ©2003 by American Society of Hematology

Expression of p210Bcr/Abl and phosphotyrosine-containing proteins. Expression of p210Bcr/Abl and phosphotyrosine-containing proteins. Immunoblot analyses were performed with Tot2p210 cells at 3 days after transduction of control or ICSBP retrovirus. Tomohiko Tamura et al. Blood 2003;102:4547-4554 ©2003 by American Society of Hematology

Down-regulation of c-Myc expression in ICSBP-transduced cells. Down-regulation of c-Myc expression in ICSBP-transduced cells. (A) Semiquantitative RT-PCR analysis for c-Myc transcripts. (B) Wright-Giemsa stain of Tot2p210 cells transduced with retroviruses carrying hormone-binding domain of estrogen receptor (ER) or ICSBP/ER chimeric construct. Cells were either left untreated or treated with 1 μM β-estradiol (Est) for 24 hours (original magnification × 600). (C) RNA blot analysis for the c-Myc transcripts. Tot2p210 cells transduced with indicated viruses were treated with 1 μM β-estradiol. Total RNA (5 μg) from cells treated for the lengths of time was analyzed. The bottom panel indicates 28S ribosomal RNA. (D) Effect of cycloheximide (CHX) on ICSBP/ER-mediated repression of c-Myc expression. The top panel indicates RNA blot analysis; the bottom panel, quantification of the c-Myc transcripts. CHX (10 μg/mL) was added 10 minutes before addition of estradiol. The expression level in CHX + Est samples was normalized by the values in CHX alone-treated cells. Tomohiko Tamura et al. Blood 2003;102:4547-4554 ©2003 by American Society of Hematology

Induction of Blimp-1 and METS/PE1 mRNA by ICSBP Induction of Blimp-1 and METS/PE1 mRNA by ICSBP. (A) Expression of Blimp-1 and METS genes in ICSBP/ER-transduced Tot2p210 cells. Induction of Blimp-1 and METS/PE1 mRNA by ICSBP. (A) Expression of Blimp-1 and METS genes in ICSBP/ER-transduced Tot2p210 cells. RNA from cells treated with 1 μM β-estradiol for the indicated time was subjected to semiquantitative RT-PCR (for Blimp-1) and RNA blot analysis (for METS). (B) EMSA detection of DNA binding activity of Blimp-1. Nuclear extracts from ICSBP/ER-transduced Tot2p210 cells with or without estradiol treatment (10 hours) were analyzed with the PRF probe. In lanes 3 and 4, extracts from estradiol-treated cells were preincubated with anti-Blimp-1 serum, normal rabbit serum, or 100-fold excess unlabeled probe, prior to addition of labeled probe. (C) Effect of cycloheximide (CHX) on ICSBP/ER induction of Blimp-1 and METS transcripts. Cells were treated as in Figure 5D. Tomohiko Tamura et al. Blood 2003;102:4547-4554 ©2003 by American Society of Hematology

ICSBP restoration of Blimp-1 and METS expression in ICSBP-/- bone marrow cells. ICSBP restoration of Blimp-1 and METS expression in ICSBP-/- bone marrow cells. (A) Blimp-1 and METS transcript levels in wild-type and ICSBP-/- bone marrow mononuclear cells. Transcript levels were quantified by real-time RT-PCR and were normalized by GAPDH levels. (B) Blimp-1 and METS transcript levels in ICSBP-transduced bone marrow lin- cells. Bone marrow lin- cells from ICSBP-/- mice were transduced with control MSCV-puro or MSCV-ICSBP-puro retrovirus on day 1 and day 2 as described in “Materials and methods.” Tomohiko Tamura et al. Blood 2003;102:4547-4554 ©2003 by American Society of Hematology

Repression of c-Myc and growth inhibition by Blimp-1. Repression ofc-Mycand growth inhibition by Blimp-1. (A) Semiquantitative RT-PCR for ectopic Blimp-1. Tot2p210 cells were transduced with control MSCV-CD8t or MSCV-Blimp-1-CD8t. Transduced cells were purified by immunomagnetic cell sorting using anti-CD8 antibody on day 2. (B) c-Myc transcript level quantified by real-time RT-PCR. (C) Cell growth rate (fold increase in cell number) during day 2 to day 6. Data represent mean ± SD in triplicate. Tomohiko Tamura et al. Blood 2003;102:4547-4554 ©2003 by American Society of Hematology

A model for a growth inhibitory pathway activated by ICSBP in myeloid cells. A model for a growth inhibitory pathway activated by ICSBP in myeloid cells. Bcr/Abl activates c-Myc expression to stimulate cell growth ICSBP induces the Blimp-1 and METS genes, which contribute to repression of the c-Myc gene and presumably other growth control genes. Bcr/Abl may down-regulate ICSBP expression to achieve efficient mitogenic transformation in CML. Tomohiko Tamura et al. Blood 2003;102:4547-4554 ©2003 by American Society of Hematology