Dysregulation of Nuclear Factor of Activated T-Cells (NFAT1) in Human T-Cell Acute Lymphocytic Leukemia (T-ALL): Possible Role of Inactivated NFAT1 in Leukemogenesis.

Abstract Introduction: NFAT1 is a transcription factor integral for the regulation of T-cell proliferation, differentiation, and apoptosis. NFAT1 is present in the cytoplasm of resting T-cells and upon stimulation (ionomycin, antigen or anti-CD3) is dephosphorylated via calcineurin and translocates to the nucleus to associate with other transcription factors (AP1). We sought to determine whether NFAT1 regulation in T-cells contributes to leukemogenesis. Initial in vitro analyses incorporated several T-ALL cell lines all derived from the peripheral blood of patients including 3 mature T-cell lines: CCRF-CEM (no clear chromosomal abnormalities), Jurkat (karyotype 46, XY, −2, −18, del(2) (p21p23), del(18) (p11.2)) Loucy (translocation t(16;20)(p12;q13), and p53 overexpression), and one immature T-cell line Molt-4 (hypertetraploid chromosomes and 6q-, t(7;7)) (ATCC Manassas, VA). Methods: T-ALL cell lines were cultured in accordance with ATCC guidelines. Surface marker analysis for CD34, CD38, HLA-DR, CD3, CD4, CD8, CD2, and CD7 was performed (BD Biosciences). Cytoplasmic and nuclear extracts were prepared from cell lines and control adult blood (AB) CD3+ cells. Cell lysates (20μg) were examined by Western blot. Blots were probed with anti-NFAT1 antibody (BD Biosciences) and protein loading controls β2-microglobulin (Abcam) and lamin A/C (Santa Cruz Biotechnology). Bands were visualized using Supersignal West Pico chemiluminescent substrate (Pierce) and quantitated using NIH imageJ software. Apoptotic assays were performed by treating samples with 0.832 μM cyclosporin A (CsA) for 48 hrs, then determining the percentage of early apoptotic cells using an Annexin V/Propidium iodide flow cytometric kit (Calbiochem). Results: Surface marker analysis confirmed that Loucy, Jurkat, and CCRF-CEM cells possess a mature phenotype, whereas Molt-4 cells possess a more immature phenotype (Table 1). Western blot analysis showed Loucy cells were unique in that NFAT1 was absent in the nuclear fraction, indicating NFAT1 is only present in it's inactive form in this cell line. Among the T-ALL cell lines, only Loucy showed no difference in the proportion of apoptotic cells following CsA treatment. Table 1 . Phenotype Analysis of T-ALL Cell Lines Surface Antigens CCRF-CEM JURKAT LOUCY MOLT-4 CD2 83.97 96.16 4.96 91.53 CD7 99.74 87.84 99.96 57.88 CD3 18.22 52.15 96.48 8.33 CD4 62.42 54.56 12.62 42.64 CD8 97.42 5.32 0.81 83.19 CD34 14.80 2.62 0.65 55.78 CD38 93.59 89.40 92.92 99.66 HLA-DR 2.31 2.62 0.56 0.33 Percentage of Total Cells Conclusions: Despite Loucy cells' mature phenotype, they exhibited distinct NFAT1 dysregulation compared to other mature cell lines. CsA treatment of Loucy cells failed to effect NFAT1 translocation to the nucleus and induction of apoptosis, as NFAT1 remained in the cytoplasm. Loucy's insensitivity to CsA with regards to apoptosis signaling supports the idea for a role for NFAT1 in Ca(2+) signaling cascade for apoptosis in T-cells. These results also suggest a possible role of cytosolic NFAT1 in leukemiogenesis, since the amount of protein seen in whole cell extracts between the various cell lines was nearly the same. The translocation in Loucy cells is in the same chromosomal location as the NFAT1 gene locus. Studies are ongoing to understand the role of NFAT1 in T-ALL initiation and progression.