ABSTRACT
The stress-inducible ULBP1 cell surface ligand for the activating immunoreceptor NKG2D allows recognition and lysis of tumor cells by natural killer (NK) and T cells. Understanding of mechanisms regulating ULBP1 expression is limited, but it is important for exploiting NKG2D-dependent antitumor responses. We studied the role of 3' untranslated region (3' UTR) in post-transcriptional regulation of ULBP1 expression in Jurkat and HeLa cells. Analysis of 2.4 kb-long 3' UTR revealed the presence of four AU-rich elements (ARE) and more then 200 putative microRNA binding sites. Stable or transient delivery of luciferase reporter constructs containing ULBP1-3' UTR sequences resulted in a strong reduction of luciferase activity to 7-22% with the full-length 3' UTR or 19%-62% with its fragments, indicating a contribution of 3' UTR to regulation of ULBP1 gene. Mutations introduced to ARE motifs significantly diminished luciferase activity, suggesting mRNA stabilizing effect of ARE. Among ULBP1-specific candidate microRNAs, we found miR-140-5p/-409-3p/-433-3p/-650 expressed in HeLa and Jurkat cells, and the microRNA involvement was supported by luciferase reporter assays with constructs carrying seed sequence mutations. However, microRNA overexpression or partial silencing of the microRNA processing enzyme Drosha did not equivocally clarify the role of microRNAs in regulation of ULBP1. Altogether these results provide evidence for a novel 3' UTR-mediated mechanism of regulation of ULBP1 at the post-transcriptional level.
Subject(s)
3' Untranslated Regions , Intracellular Signaling Peptides and Proteins/metabolism , Killer Cells, Natural/metabolism , Luciferases/metabolism , Regulatory Sequences, Ribonucleic Acid , 3' Untranslated Regions/genetics , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , Gene Expression Regulation, Neoplastic , Genes, Reporter/genetics , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Jurkat Cells , Killer Cells, Natural/pathology , Ligands , Luciferases/genetics , MicroRNAs/genetics , Mutagenesis, Site-Directed , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Protein Processing, Post-Translational , Regulatory Sequences, Ribonucleic Acid/geneticsABSTRACT
This study exploited alloreactivity of natural killer (NK) cells for augmenting the recognition of human acute myeloid leukemia (AML). To circumvent the inhibitory effect of killer immunoglobulin receptor (KIR) signaling, we generated NK-cell lines with single KIR specificities for major human leukocyte antigen (HLA) class I allotypes. We demonstrated efficient cytolysis of KIR-HLA class I-mismatched primary AML blasts even at low effector-to-target ratios. To define the impact of tumor-associated activating NKG2D-ligands (NKG2D-L), 66 AML patients at diagnosis were analyzed. NKG2D-L were selectively expressed on monoblastic cells in AML M4 and M5 yet absent or weakly expressed on myeloblastic cells in all AML subtypes. Paucity of cell-surface NKG2D-L was not the result of shedding because levels of soluble ULBP1 ligand measured in AML plasma were in the normal range. Notably, purified NKG2D-L(+) monoblastic cells were more susceptible to NK-mediated killing than NKG2D-L(-) myeloblastic cells. Accordingly, induction of cell-surface NKG2D-L by treatment with the histone deacetylase inhibitor, valproic acid, rendered cells more sensitive to NK cytolysis. These data suggest that adoptive transfer of selected populations of alloreactive HLA class I-mismatched NK cells in combination with pharmacologic induction of NKG2D-L merits clinical evaluation as novel approaches to immunotherapy of human AML.
