Transcriptional silencing of the ETS1 oncogene contributes to human granulocytic differentiation.
BACKGROUND
Ets-1 is a widely expressed transcription factor implicated in several biological processes including hematopoiesis, where it contributes to the regulation of cellular differentiation. The functions of Ets-1 are regulated by transcription factors as well as by phosphorylation events: phosphorylation of threonine 38 activates Ets-1, whereas phosphorylation of a cluster of serines within exon VII reduces DNA binding activity. This study focuses on the role of Ets-1 during granulocytic differentiation of NB4 promyelocytic and HL60 myeloblastic leukemia cell lines induced by all-trans retinoic acid.


METHODS
Ets-1 expression was measured by real-time reverse transcriptase polymerase chain reaction and western blotting. The role of Ets-1 during all-trans retinoic acid-induced differentiation was analyzed by using a transdominant negative molecule or small interfering RNA.


RESULTS
NB4 and HL60 cell lines expressed high levels of p51 Ets-1, while the splice variant isoform that lacks exon VII (p42) was almost undetectable. The addition of all-trans retinoic acid reduced p51 Ets-1 levels and induced inhibitory phosphorylation of the remaining protein. Expression of Ets-1 was also reduced during dimethylsulfoxide-induced differentiation and during granulocytic differentiation of human CD34(+) hematopoietic progenitor cells but not in NB4.R2 and HL60R cells resistant to all-trans retinoic acid. In line with these observations, transduction of a transdominant negative molecule of Ets-1, which inhibited DNA binding and transcriptional activity of the wild-type Ets-1, significantly increased chemical-induced differentiation. Consistently, Ets-1 knockdown by small interfering RNA increased the number of mature neutrophils upon addition of all-trans retinoic acid. Interestingly, p51 Ets-1 over-expression was frequently observed in CD34(+) hematopoietic progenitor cells derived from patients with acute myeloid leukemia, as compared to its expression in normal CD34(+) cells.


CONCLUSIONS
Our results indicated that a decreased expression of Ets-1 protein generalizes to granulocytic differentiation and may represent a crucial event for granulocytic maturation.
Lulli
Valentina
V
Dept. of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, viale Regina Elena 299, Rome, Italy.
Romania
Paolo
P
Riccioni
Roberta
R
Boe
Alessandra
A
Lo-Coco
Francesco
F
Testa
Ugo
U
Marziali
Giovanna
G
D002454:Cell Differentiation / Q000187:drug effects*; D045744:Cell Line, Tumor; D020868:Gene Silencing / Q000187:drug effects*; D006098:Granulocytes / Q000166:cytology / Q000187:drug effects*; D018922:HL-60 Cells; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects; D006801:Humans; D007938:Leukemia / Q000473:pathology; D015473:Leukemia, Promyelocytic, Acute; D050784:Proto-Oncogene Protein c-ets-1 / Q000235:genetics* / Q000502:physiology; D034741:RNA, Small Interfering / Q000494:pharmacology
D050784:Proto-Oncogene Protein c-ets-1; D034741:RNA, Small Interfering