Abstract A61: Retinoblastoma protein-interacting zinc finger 1 (RIZ1) regulates proliferation of monocytic leukemia cells in response to tumor necrosis factor (TNF)-α via activation of p53
Authors: Noman Abu Shadat M. , Koide Naoki , Khuda Imtiaz I.-E. , Dagvadorj Jargalsaikhan , Tumurkhuu Gantsetseg , Naiki Yoshikazu , Komatsu Takayuki , Yoshida Tomoaki , Yokochi Takashi 
Published: May 2012

Purpose of the Study: Retinoblastoma protein-interacting zinc finger 1 (RIZ1) is a tumor suppressor whose inactivation plays an important role in several human cancers including leukemia, osteosarcoma and breast cancer whereas the forced expression of RIZ1 inhibits tumor progression. But how the expression of RIZ1 inhibits tumor cell growth and the signaling molecules involved in this mechanism is not clear yet. The implication of inflammatory mechanisms in regulating cancer is current concern. Tumor necrosis factor (TNF)-α is an inflammatory cytokine which potentially regulates tumor microenvironment especially in myeloma and leukemia. The present study was designed to understand the regulatory action of RIZ1 on the cell proliferation of monocytic leukemia cells in response to TNF-α that is yet to characterize.

Experimental Procedures: Mouse and human monocytic leukemia cells RAW 267.4 and U937 were stimulated with TNF-α at different dose and time dependent manner and the expression of RIZ1 were analyzed by immunoblotting and RT-PCR, respectively. To clarify the signal molecules triggering TNF-α-induced RIZ1 expression, cells were treated with pharmacological inhibitors or transfected with dominant negative mutants. To investigate the RIZ1 and p53 interaction, cells were transfected with RIZ1-specific (400 nM) and nonspecific siRNA and immunoblot with anti p53 antibody. RIZ1 was immunoprecipitated by anti-RIZ1 antibody and p53 was analyzed by immunoblotting. The effect of p53 inhibitor on TNF-α-induced RIZ1 expression was examined to check the role of p53 on RIZ1 expression. The effect of RIZ1 siRNA on the proliferation of TNF-α-treated cells were examined by a cell proliferation assay using BrdU and further confirmed by the expression of PCNA as a cell proliferation marker. To assess the anchorage independent growth, a soft agar colony assay was performed.

Results: RIZ1 was significantly induced by TNF-α in RAW 264.7 and U937 cells in time and dose dependently. TNF-α induced RIZ1 expression was clearly suppressed by NF-κB and AKT dominant negative plasmid as well as pharmacological inhibitors. RIZ1 siRNA inhibited the activation of p53 in TNF-α-treated cells. The presence of p53 in the RIZ1 fraction precipitated by anti-RIZ1 antibody suggests the formation of RIZ1/p53 complex. Further, augmentation of RIZ1 expression by p53 inhibitor indicates the negative feedback by p53. The expression of PCNA and cell growth in TNF-α-treated monocytic leukemia cells were increased in RIZ1 silenced cells. The down-regulation of RIZ1 with the siRNA enhanced the soft agar colony formation in TNF-α-treated cells. Conclusion: RIZ1 was induced by TNF-α and the expression was dependent on NF-κB and AKT signaling. Further, silencing of RIZ1 prevented p53 activation and augmented the proliferative activity of TNF-α-treated cells. Here, we demonstrate that RIZ1 negatively regulates the proliferation of TNF-α treated monocytic leukemia cells via activation of p53. The RIZ1-p53 interaction might explore many questions to understand tumor suppressive mechanism of p53.

Citation Information: Cancer Res 2009;69(23 Suppl):A61.