ABSTRACT
PCs are responsible for the production and secretion of antibodies, the effector molecules of the humoral immune response. The molecular mechanisms responsible for vesicle docking and secretion implicated in the antibody-secretion process are not well-known, as they have not been studied, but it is known that SNARE proteins are responsible for many membrane-fusion processes in the cell. We show here that freshly isolated human colon LP-PCs and T-PCs from MM-PC patients and the U266 cell line, as a model for PC secretion, contain a set of these proteins. SNAP23, STX3, and STX4 were localized mainly in the plasma membrane of PCs, and interactions of SNAP23 with STX3 and with STX4 were proven by IP. Interaction between SNAP23 and STX4 was also confirmed in situ. With the use of siRNA, as well as shRNA, the functional role of SNAP23, STX3, and STX4 in antibody secretion was also examined. The findings demonstrate that in addition to SNAP23, STX4 is implicated in the antibody secretion by a myeloma cell line and by normal human colon LP-PCs.
Subject(s)
Antibodies/metabolism , Plasma Cells/metabolism , Qa-SNARE Proteins/metabolism , Cell Line , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Separation , Doxycycline/pharmacology , Humans , Immunoglobulin A/biosynthesis , Immunoglobulin E/metabolism , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Plasma Cells/drug effects , Protein Binding/drug effects , Protein Transport/drug effects , Qb-SNARE Proteins/metabolism , Qc-SNARE Proteins/metabolism , RNA, Small Interfering/metabolism , Subcellular Fractions/drug effects , Subcellular Fractions/metabolismABSTRACT
The positive regulatory domain containing 1, encoded by the PRDM1 gene, is a transcriptional repressor considered as a master regulator that is required and sufficient for plasma cell differentiation. In the present study we have performed sequence analysis of the upstream region of the human PRDM1 gene to detect the minimal promoter region necessary for PRDM1 gene transcription. This region comprises the region upstream of the initiation site, as well as the first exon. Collectively, deletion and mutation analysis in conjunction with luciferase reporter assays, EMSA and supershift assays identified a phylogenetically conserved GC-box as an essential element for PRDM1 expression. This GC-box element matches to a binding site for multiple transcription factors such as SP1 and SP3 isoforms as well as early growth response 1. Chromatin immunoprecipitation assays confirmed the in vivo binding capability of these factors to the human PRDM1 promoter. These studies together characterize for the first time the basal activity of the human PRDM1 promoter, through which several factors, including SP1, SP3 and early growth response 1, modulate its expression through a conserved GC-box.
Subject(s)
Early Growth Response Protein 1/physiology , Repressor Proteins/genetics , Sp1 Transcription Factor/physiology , Sp3 Transcription Factor/physiology , Transcription, Genetic , 5' Flanking Region , Base Sequence , Cell Differentiation , Cells, Cultured , Humans , Ionomycin/pharmacology , Molecular Sequence Data , Positive Regulatory Domain I-Binding Factor 1 , Promoter Regions, Genetic , Regulatory Elements, Transcriptional , Tetradecanoylphorbol Acetate/pharmacology , Transcription Initiation SiteABSTRACT
BACKGROUND AND OBJECTIVES: Chemokine receptors are involved in tumor progression and several of these receptors, including CXCR3, are expressed by chronic lymphocytic leukemia (CLL) B cells. This study was aimed to examine a possible relationship between CXCR3 expression in CLL and the clinical evolution of the disease. DESIGN AND METHODS: Using flow activated cell sorting (FACS), we analyzed the level of expression of CXCR3 on blood CLL B cells from 76 consecutive patients. The results were correlated with CD38 expression, IgVH gene status and clinical outcome. RESULTS: CXCR3, measured as mean fluorescence intensity (MFI), was unimodally expressed by blood tumor cells at various levels (range, 3.5 to 232.3) but levels within individual patients were remarkably stable over time. Low CXCR3 expression by CLL B cells was strongly associated with Rai disease stages III and IV (p<0.0001) and a pattern of diffuse tumor infiltration of the bone marrow (p<0.0001). In the 28 cases available for genetic studies, low CXCR3 expression also showed good concordance with tumor unmutated IgVH gene status (p<0.04), and tended to correlate with high CD38 expression (p<0.06). Patients with low CXCR3 expression (MFI < or =15) had a shorter survival (p<0.0001) and, in multivariate analysis, low CXCR3 expression (MFI pound15) was an independent predictor of poor outcome (hazard ratio 24.5; p<0.01). INTERPRETATION AND CONCLUSIONSL: CXCR3 expression by CLL B cells appears to be stable within individual patients. Tests to assay this chemokine receptor are cheap and easy to perform and the results could be of prognostic value in CLL.
