CDDO Increases Translation of CCAAT Enhancer Binding Protein alpha To Induce Granulocytic Differentiation.

Abstract

The triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) is a novel antineoplastic drug which induces apoptosis of a wide variety of tumor cells in vitro and in vivo and leads to granulocytic differentiation of hematopoietic progenitor cells. We studied the effect of CDDO on CCAAT enhancer binding protein alpha (CEBPA), a transcription factor which is critical for granulocytic differentiation. In HL60 myeloblastic cells, CDDO (0.01 to 2 uM) dose-dependently decreased the number of cells in culture and increased the fraction of apoptotic cells. However, at doses which did not induce apoptosis, CDDO increased the number of granulocytic cells, as assessed by morphology, NBT assay, and FACS, and Northern blotting showed an increase of GCSFR and a decrease of c-myc mRNA. Phagocytosis of FITC-labeled E. coli bacteria by these cells was enhanced by CDDO. While CEBPA mRNA was decreased, CEBPA protein was significantly increased within 24 hours of treatment, and this was not abrogated by preincubation with the caspase inhibitor Z-DEVD-fmk, again suggesting that these effects were independent of apoptosis. CDDO increased the ratio of the transcriptionally active isoform p42 and the inactive p30 isoform 3-fold, and gel shift assays showed enhanced DNA binding to a GCSFR promoter probe. Since eukaryotic translation initiation factors (eIF) have been described to alter the CEBPA protein isoform ratio, we studied the effects of CDDO on eiF2 alpha and eiF4E activity. CDDO increased the phosphorylation of eIF4E and decreased the phosphorylation of eIF2 alpha within 5 hours of treatment, and this was associated with an increase of the p42/p30 CEBPA ratio. In the presence of the translation inhibitor cycloheximide, CEBPA protein levels decreased after 2 hours, suggesting that CDDO did not stabilize CEBPA and that de novo protein synthesis was required for the observed effects. The effect of CDDO on the p42/p30 ratio was mimicked by 2-AP, which inhibits eIF2 alpha phosphorylation, but was independent of PPARgamma and TGFß pathways, as demonstrated by preincubation with GW9662, or TGFß1, respectively. In primary blasts from patients with acute myeloid leukemia (AML), the p42/p30 ratio of CEBPA was enhanced by CDDO treatment. In conclusion, CDDO leads to granulocytic differentiation and translational induction of CEBPA protein. Since CEBPA function is impaired in many patients with AML, CDDO may provide a novel treatment approach for these patients.