ABSTRACT
Oncostatin-M (OSM) is a patho-physiologically important pleiotropic, multifunctional cytokine. OSM mRNA sequence analysis revealed that its 3'UTR contains three highly conserved GC-rich cis-elements (GCREs) whose role in mRNA stability is unidentified. In the present study, the functional role of the proximal GC-rich region of osm 3'-UTR (GCRE-1) in post-transcriptional regulation of osm expression in U937 cells was assessed by transfecting construct containing GCRE-1 at 3'-end of a fairly stable reporter gene followed by analysis of the expression of the reporter. GCRE-1 showed mRNA destabilizing activity; however, upon PMA treatment the reporter message containing GCRE-1 was stabilized. This stabilization is owing to a time-dependent progressive binding of trans-factors (at least five proteins) to GCRE-1 post-PMA treatment. Nucleolin was identified as one of the proteins in the RNP complex of GCRE-1 with PMA-treated U937 cytosolic extracts by oligo-dT affinity chromatography of poly-adenylated GCRE-1. Immuno-blot revealed time-dependent enhancement of nucleolin in the cytoplasm which in turn directly binds GCRE-1. RNA co-immunoprecipitation confirmed the GCRE-1-nucleolin interaction in vivo. To elucidate the functional role of nucleolin in stabilization of osm mRNA, nucleolin was overexpressed in U937 cells and found to stabilize the intrinsic osm mRNA, where co-transfection with the reporter containing GCRE-1 confirms the role of GCRE-1 in stabilization of the reporter mRNA. Thus, in conclusion, the results asserted that PMA treatment in U937 cells leads to cytoplasmic translocation of nucleolin that directly binds GCRE-1, one of the major GC-rich instability elements, thereby stabilizing the osm mRNA.
Subject(s)
3' Untranslated Regions , Monocytes/metabolism , Oncostatin M/genetics , Phosphoproteins/genetics , RNA Stability , RNA-Binding Proteins/genetics , Base Composition , Binding Sites , Cell Line, Tumor , Gene Expression Regulation , Humans , Monocytes/cytology , Monocytes/drug effects , Oncostatin M/metabolism , Phosphoproteins/metabolism , Plasmids/chemistry , Plasmids/metabolism , Protein Binding , RNA-Binding Proteins/metabolism , Signal Transduction , Tetradecanoylphorbol Acetate/pharmacology , Transfection , NucleolinABSTRACT
Diallyl disulfide (DADS), the major organosulfur component of processed garlic is very effective in chemoprevention of several types of cancers; however, its detailed mechanism is yet to be divulged. Present study shows antiproliferative activity of DADS against human leukemic cell-lines, mainly U937. DADS induced transient G2/M phase arrest, which is evident from FACS analysis. The results revealed that a significant transcriptional induction of p21 happened in early hours of treatment, which is due to increased nuclear translocation of NF-κB and its specific binding to p21 promoter. However, in the later hours, G2/M arrest is lost leading to apoptosis via intrinsic mitochondria-mediated pathway through generation of reactive oxygen species followed by changes in mitochondrial membrane potential. Western blots indicate release of cytochrome-c, activation of caspase-3, cleavage of PARP1, and finally decrease in bcl-2 levels. In addition, inactivation of NF-κB by its inhibitor BAY 11-7085 causes early onset of apoptosis without any transient G2/M arrest. Thus, in conclusion, DADS induces reversible G2/M arrest through NF-κB mediated pathway in human leukemic cell lines, like U937, K562, and Jurkat, lacking wild type p53. However, G2/M arrest is lost owing to the incapability of the damage repair system that leads to apoptosis.
