ICAT as a potential enhancer of monocytic differentiation: implications from the comparative proteome analysis of the HL60 cell line stimulated by all‐trans retinoic acid and NSC67657

Abstract

A novel sterol mesylate compound (NSC67657) was recently identified and reported by National Cancer Institute that could efficiently induce the differentiation of HL60 cells into monocytes in vitro and in vivo. The expression of many proteins would have been changed during the differentiation process, and some proteins may have played key roles in the differentiation of HL60 cell line induced by this drug. Therefore, we treated HL60 cells with NSC67657 and all-trans retinoic acid (ATRA) to identify the differentially expressed proteins and determine their functions in cellular differentiation. Of the 45 differentially expressed protein spots investigated, 24 were either elevated or decreased in both the monocytic and granulocytic differentiating HL60 cells, 8 showed significant changes only when induced by NSC67657, and 13 showed significant changes only when induced by ATRA. After verification by RT-PCR, Western blotting, and immunocytochemistry, only the protein ICAT was found to be elevated by NSC67657 treatment alone. Although the over-expression of ICAT is not sufficient to induce the differentiation of HL60 cells into monocytes, it did increase the proportion of CD14+ cells in cells pretreated with NSC67657. Successful application of multiple techniques including two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, Western blotting, and eukaryotic electroporation revealed that proteomic and molecular biological analyses provide valuable tools in drug development research.

Expression level and prognostic potential of beta-catenin–interacting protein in acute myeloid leukemia

“…NSC67657 is significantly inhibited if the gene encoding intracellular ICAT is silenced. [ 21 ] It has been suggested in a few studies [ 8 ] that the expression level of intracellular ICAT protein level can interfere with chemotherapy and may represent a tumor suppressor in leukemia cells. Although ICAT is a differential protein expressed specifically during monocyte differentiation in HL60 cells and plays an important role in monocyte differentiation, [ 8 ] its regulatory role may interfere with granulocyte induction chemotherapy.…”

“…Thus, we observed that catenin, beta-interacting protein 1 (inhibitor of beta-catenin and TCF [ ICAT ] or CTNNBIP1 ), an inhibitor of β-catenin is upregulated during monocyte differentiation, can enhance drug sensitivity of HL60 cells. [ 8 ] The ICAT molecule plays a crucial role in the Wnt/β-catenin signaling pathway, and in a competitive region with T-cell factor ( TCF )/lymphocyte enhancement factor ( LEF ), binds to the tandem protein and E-cadherin and inhibits Wnt signal transduction as well as intracellular adhesion, [ 9 , 10 ] which plays a critical role in tumorigenesis. Therefore, it has been observed in several studies that ICAT acts as a caner suppressor.…”

Opposing roles of ICAT and Wnt/β-catenin signaling in NSC67657-induced monocytic differentiation

“…NSC67657 is a highly efficient inducer of monocytic differentiation [ 4 ]. The NSC67657- induced ICAT levels in HL60 cells suggested a link between the tumor suppressive effects of NSC67657 and deregulated Wnt signaling [ 5 , 6 ]. Mutations involving components of the Wnt signaling cascade, especially in APC or β-catenin, are essential for initiation of many cancers, including colorectal cancer [ 16 ].…”

“…All-trans retinoic acid and NSC67657 have been used to induce differentiation of HL60 cells to granulocytes and monocytes, respectively. Comparative proteomics studies identified the differentiation-specific protein ICAT and demonstrated its increased expression in differentiated HL60 monocytic cells [ 5 ]. ICAT is an 81 amino acid protein previously identified in a yeast two-hybrid screen, using the armadillo repeat region of β-catenin as a bait.…”

A20 is up‐regulated in primary mouse hepatocytes subjected to hypoxia and reperfusion

“…The proteomics approach offers a powerful methodology to evaluate the global alterations in biological systems at the protein level and has been successfully applied to reveal the global proteomic profiling of cells and animal models under different physiopathological conditions . Unfortunately, the successful application of proteomics to identify ischemia reperfusion–induced protein changes in the hepatic tissue remains a technical challenge because of the cellular heterogeneity of the hepatic tissue.…”