CCAAT/enhancer binding protein ε is a potential retinoid target gene in acute promyelocytic leukemia treatment
Dorothy J. Park, Alexey M. Chumakov, Peter T. Vuong, Doris Y. Chih, Adrian F. Gombart, Wilson H. Miller Jr., H. Phillip Koeffler
Dorothy J. Park, Alexey M. Chumakov, Peter T. Vuong, Doris Y. Chih, Adrian F. Gombart, Wilson H. Miller Jr., H. Phillip Koeffler
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CCAAT/enhancer binding protein ε is a potential retinoid target gene in acute promyelocytic leukemia treatment

Abstract

The CCAAT/enhancer binding protein ε (C/EBPε) is a nuclear transcription factor expressed predominantly in myeloid cells and implicated as a potential regulator of myeloid differentiation. We show that it was rapidly induced in the acute promyelocytic leukemia (APL) cell line NB4 during granulocytic differentiation after exposure to retinoic acid (RA). Our data suggest that induction of C/EBPε expression was through the retinoic acid receptor α (RARα) pathway. Reporter gene studies showed that C/EBPε promoter/enhancer activity increased in a retinoid-dependent fashion via the retinoic acid response element (RARE) present in the promoter region of C/EBPε. The RA-induced expression of C/EBPε markedly increased in U937 myelomonoblasts that were induced to express promyelocytic leukemia/RARα (PML/RARα), but not in those induced to express promyelocytic leukemia zinc finger/RARα (PLZF/RARα). In retinoid-resistant APL cell lines, C/EBPε either is not induced or is induced only at very high concentrations of RA (≥10–6 M). In addition, forced expression of C/EBPε in the U937 myelomonoblastic leukemia cells mimicked terminal granulocytic differentiation, including morphologic changes, increased CD11b/CD66b expression, and induction of secondary granule protein expression. Our data strongly suggest that C/EBPε is a downstream target gene responsible for RA-induced granulocytic differentiation of APL cells.

Authors

Dorothy J. Park, Alexey M. Chumakov, Peter T. Vuong, Doris Y. Chih, Adrian F. Gombart, Wilson H. Miller Jr., H. Phillip Koeffler

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Transactivational abilities of C/EBPε promoter/enhancer. (a) Schema of C...
Transactivational abilities of C/EBPε promoter/enhancer. (a) Schema of CAT reporter constructs with putative retinoic acid response element from C/EBPε (RAREC/EBPε), mutant RAREC/EBPε, 0.5-kb C/EBPε promoter/enhancer region, and mutant 0.5-kb C/EBPε promoter/enhancer region. (b) Retinoid-induced transactivation of C/EBPε promoter/enhancer in COS-1 cells. The transactivation of C/EBPε promoter/enhancer CAT reporters by RARα, mutant RARα (RARα403), and PML/RARα were compared in either the presence (hatched bars) or absence (solid bars) of ATRA (10–6 M). The -fold induction of transactivation is calculated relative to that of pCMX transfected without ATRA treatment. (c) Retinoid-dependent transactivation of C/EBPε promoter/enhancer in the myeloid leukemia cell line KCL22. (d) Retinoid-dependent transcriptional activation of RAREC/EBPε-tk-CAT (CAT reporter containing 3 copies of putative RARE found in C/EBPε promoter/enhancer region in tandem repeats upstream of a minimal tk promoter) in COS-1 cells. (e) Lack of retinoid-dependent transcriptional activation by mut-p0.5-tk-CAT in COS-1 cells. (f) DNA binding and EMSA of the RAR/RXR to RAREC/EBPε. The 32P-labeled RAREC/EBPε oligonucleotide was used as a probe for DNA binding and EMSA with a GST-RAR, GST-RXR, or GST control. For competition, either unlabeled wild-type or mutant RAREC/EBPε oligonucleotides in increasing amounts (1-, 10-, or 100-fold excess) were used in the binding reaction with the 32P-radiolabeled RAREC/EBPε and RAR/RXR heterodimer. Bands a and b represent either RAR/RAR homodimers or RAR/RXR heterodimers bound to RAREC/EBPε oligonucleotide probe. Band c represents unbound probe.