Abstract 1006

Poster Board I-28

Onzin is a small, novel and highly conserved cysteine-rich protein with unique structure and tissue-restricted expression, at the high levels in myeloid, intestine and spleen as well as at lower level in lung. Previous reports indicate that inhibition of endogenous onzin results in a reduced growth rate and an increased sensitivity to apoptotic stimuli, while the enforced expression of onzin in fibroblasts leads to marked apoptotic resistance, loss of G2/M checkpoint control and tumorigenic conversion. However, both the regulation of its expression and its biological role remain greatly elusive. In this study, we provided the first demonstration that onzin expression is significantly downregulated during differentiation induction of acute myeloid leukemic (AML) cell lines and primary cells from AML patients by phorbol 12-myristate 13-acetate (PMA) and all-trans retinoic acid (ATRA). Further, this suppression of onzin expression can also be found in PMA-treated solid tumor cell lines with physiological onzin expression by screening 9 different tumor cells. To explore the potential mechanism of onzin downregulation, next we used PMA as the probe in the following experiment. Applying chemical selective inhibitors and transfected expression of dominant negative mutants or constitutive catalytic form of the related kinases, we showed that Protein kinase C-epsilon (PKCε)-extracellular signal-regulated protein kinase (ERK) signaling axis is required for PMA-induced downregulation of onzin expression. Then, to investigate whether the downregulation of onzin expression is associated with leukemic cell differentiation, the full-length onzin cDNA together with empty vector as a control were respectively transfected into NB4 cells as well as U937 cells by retrovirus infection. NB4 and U937 cells with onzin infection expressed higher level of onzin protein could sufficiently overcome the decrease of endogenous onzin protein induced by PMA. Compared with NB4 and U937 cells with empty vector infection, interestingly, onzin overexpression inhibited PMA- or ATRA-induced cell differentiation, evidenced by CD11b and CD14 expression, NBT assays and morphologic alterations. Finally, to exploit how onzin affects PMA- and ATRA-induced differentiation, we investigated whether onzin interacts with the Runt-related protein 1(Runx1), CCAAT/enhancer-binding protein α (C/EBPα) and PU.1, three important transcriptional factors in hematological cell differentiation. CO-IP results indicate that onzin can effectively interact with PU.1 in 293T and U937 cells, in which the two proteins are respectively ectopically expressed and endogenously expressed, but onzin failed to interact with Runx1 or C/EBPα. Furthermore, we examined the effect of onzin on pM-CSFR-luc (driven by human macrophage colony-stimulating factor receptor promoter containing Runx1, C/EBPα and PU.1 binding sites) activity when transiently transfected with Runx1/CBFα, C/EBPα or PU.1 and onzin alone or together in 293T cells. The results showed that although onzin transefected alone had no effect on M-CSFR-luc activity, onzin can partially but significantly decreased M-CSFR-luc activity driven by PU.1, while the forced expression of onzin also had no effect on the M-CSFR-luc activity driven by Runx1 and C/EBPα. Taken together, this is the first report demonstrating that PMA and ATRA downregulates onzin expression through PKCε-ERK signaling pathway, which favors leukemic cell differentiation.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.