MYL9 Is Prerequisite for the CD3 Expression in Cytoplasm Membrane of T Lymphocytes

Abstract

Abstract 4925 [Introduction]Normal T lymphocytes express CD3 protein, major component of T-cell receptor, in the cytoplasm membrane. However, CD3 expression is suppressed in malignant T cells. The mechanism of suppression of CD3 expression remains unknown. We previously reported the cloning of three isoforms of non-muscle myosin light chain, including MYL9 (transcript variant 1). MYL9 mRNA was expressed in monocyte-macrophage cell lines, but not in lymphoid cell lines (J. Smooth Muscle Res.44: 29–40, 2008). We report here some evidence, suggesting the prerequisite role of MYL9 (transcript variant 1) in the process of CD3 expression in cytoplasm membrane. [Materials and Methods]Leukemic cell lines were cultured in RPMI1640 plus 10% FCS. mRNA contents were measured with RT-PCR. CD3 expression was measured using FACScan FITC-labeled or PE-labeled second antibody. Whole CD3 contents were measured using IntraPrep™ (Beckman Coulter). Transfection of MYL9(transcript variant 1)gene into Jurkat cell (wild-type) was performed using Trans Fasc Reagent (Promega). In the process of screening the change the percentage of surface marker, we found that some clones showed the CD3 positive, and Jurkat (clone I), which expresses CD3 (85∼90%) in the cytoplasm membrane, was selected. We also used Jurkat, Clone E6-1(TIB-152)(ATCC®), strong positive in CD3 (90∼95%) in cytoplasm membrane. [Results]1) CD3 were negative in Jurkat cell (wild-type), and highly positive in both clone I and clone E6-1. 2) Whole CD3 (cytosole and cytoplasm membrane) were positive in these three cells, suggesting that transportation of CD3 protein from cytosole to cytoplasm membrane is suppressed in Jurkat cell (wild-type). 3) Long term culture (i.e. 30∼60 days) induced, although weakly, both MYL9 mRNA and CD3 of cytoplasm membrane in Jurkat cell (wild-type). 4) Clone E6-1 shows positive in MYL9 mRNA. 5) In Jurkat cell (wild-type), PMA induced CD3 expression in cytoplasm membrane, but not MYL9 mRNA. Pre-treatment with staurosporine showed partial inhibitory effect on PMA-induced CD3 expression. 6) Neither concanavalin A nor A23187 induced CD3 expression in both clone I and Jurkat cell (wild-type). [Discussion]These results suggest that MYL9 is prerequisite for the CD3 expression in cytoplasm membrane of normal T lymphocytes. However, in T-cell malignant cells, MYL9 gene is suppressed by unknown mechanism. Our data show that CD3 expression in cytoplasm membrane may be regulated by MYL9-dependent and MYL9-independent fashions. Disclosures:No relevant conflicts of interest to declare.