Nuclear Factor of Activated T-Cells (NFAT1): Novel Target In T-Cell Acute Lymphocytic Leukemia (T-ALL)

The well-characterized human T-cell transcription factor NFAT1 has been reported to regulate cell cycle regulatory proteins including Cyclins A, D, and p21. This group has recently identified microRNA-184 to regulate NFAT1 protein translation in human umbilical cord blood CD4 T-cells. We sought to determine if NFAT1 protein expression is dysregulated in T-ALL, and if so, its contribution, if any to T-ALL pathogenesis.

MOLT-4 cells were maintained according to ATCC guidelines. Normal adult and umbilical cord blood (UCB) T-cells were selected by AutoMACS (Miltenyi Biotec) and and maintained per standard protocols. Purity was confirmed in excess of 90% by flow cytometry. MOLT-4 cells were transfected with a 10 pmol:3.33 pmol ratio of anti-miR-184 miRNA inhibitor (Ambion) to siGLO non-specific RISC-free control (Dharmacon) in complete RPMI alongside corresponding controls via lipofectamine RNAi max (Invitrogen) overnight per manufacturers’ instructions. Protein was isolated 48 hours after transfection and quantified by standard protein assay (BioRad). 10 μ g of each fraction was analyzed by 10% SDS-PAGE followed by Western Blotting with anti-NFAT1 (BD Biosciences) and γ-tubulin (Santa Cruz Biotechnology) antibodies. Bands were visualized using Supersignal West Pico chemiluminescent substrate (Pierce).

The relative levels of both NFAT1 protein and mir-184 were first compared to normal adult and UCB CD4+ T-cells. As miR-184 and NFAT1 expression levels can change over the developmental life of CD4+ T-cells, a comparison of MOLT-4 cells was made to normal UCB T-cell controls with similar surface phenotypes (CD4+/8+). NFAT1 protein expression was found to be significantly decreased in the MOLT-4 cells by Western Blot. Further, comparisons of miR-184 expression in UCB double positive T-cells to MOLT-4 by quantitative reverse transcription real-time PCR (qPCR) demonstrated a 56-fold higher expression of mir-184 in MOLT-4 compared to controls. Next, in order to test the ability of miR-184 to regulate NFAT1 levels in the MOLT-4 T-ALL cell line, lipofectamine-mediated transfections of antisense miR-184 were performed. Western Blot analysis showed the expected modest increase in the NFAT1 protein expression in MOLT-4 cells transfected with anti-miR-184.

NFAT1 protein levels are notably reduced in T-ALL cell lines and may be attributable in part to 56-fold higher expression of miR-184. Knockdown of miR-184 renders normalization of NFAT1 protein levels. Ongoing studies are designed to determine cell cycle kinetics and p21 expression in miR-184 transfected MOLT-4 cells. Dysregulation of NFAT1 may contribute to T-ALL pathogenesis, and miR-184 regulation of NFAT1 may be a target for therapeutic studies.

No relevant conflicts of interest to declare.