Expression Of Ikaros Isoforms Research Articles

Selected Adaptor Proteins NTAL, LAT, PAG, LIME Function in Proximal Signaling, Corticosteroid Driven Apoptosis and Expression of IKAROS Isoforms in the T-Leukemic Cells.

Abstract 5034Transmembrane adaptor proteins NTAL, LAT, PAG and LIME have an important role in proximal signaling of T-lymphocytes. They lack enzymatic or kinase function, but when tyrosine-phosphorylated they bind other signaling molecules and mediate signaling from T-cell receptor (TCR) to nucleus. In our previous studies, we showed that in childhood T-cell acute lymphoblastic leukemia (T-ALL) the level of NTAL significantly correlates with early response to treatment. Patients with high NTAL levels responded favorably (prednisone good-responders) whereas patients with low NTAL were prednisone poor responders (p=0.05). We confirmed this clinical data also in in-vitro experiment - derivative Jurkat cell line (human T-ALL) transfected with NTAL expression construct (Jurkat/NTAL+) was more sensitive to corticosteroid treatment compared to wild-type Jurkat cell line (Jurkat/wt, NTAL negative).In the present study, we performed a more detailed analysis of a relationship between adaptor proteins, TCR signaling and apoptosis in T-ALL. Monoclonal IgM antibody C305 binding the Jurkat TCR with high affinity was used to stimulate TCR signalization in Jurkat/wt and Jurkat/NTAL+ cell lines. To assess the influence of TCR signaling on corticosteroid-driven apoptosis, we treated both the Jurkat cell lines with the C305 antibody and/or with methylprednisolone. At 24 hours we detected higher percentage of cells with the stimulation marker CD69 in Jurkat/NTAL+ cells compared to the Jurkat/wt (median 70% vs. 60%, p<0.05). More cells treated with C305 and methylprednisolone together expressed higher CD69 than cells treated by C305 alone (median 75% vs. 65%, p<0.004). At 24 and 48 hours we determined the level of apoptosis. At each time point the number of living cells in the untreated control was set to 100%. Jurkat/NTAL+ cell line was more prone to apoptosis than the wild-type cell line in all settings. At 24 hours, the percentage of surviving cells Jurkat/NTAL+ vs. Jurkat/wt was 44% vs. 50% when treated with methylprednisolone alone (p<0.1), 24% vs. 42% (C305 alone, p<0.1) and 15% vs. 26% (methylprednisolone + C305, p<0.1). Similar differences were detected after 48hours of treatment. Using flow cytometry, we further determined phosphorylation status of key downstream kinases ERK, p38 and JNK after TCR stimulation by the C305 antibody. We detected hyperphosphorylation of ERK higher in Jurkat/NTAL+ compared to Jurkat/wt (1.57, 1.53 and 1.46 fold at 5, 15 and 30 minutes after stimulation, respectively). Levels of phosphorylated P38 and JNK did not differ from the unstimulated controls or between the two cell lines. Our data show that the differences in apoptosis are not driven by an upregulation of FAS or FAS ligand as any significant increase in the levels of FAS/FASL was not detected by flow cytometry throughout the above described experiments.It was shown previously that aberrant TCR signaling affects the incidence of dominant-negative isoforms of IKAROS. Thus, to further elucidate the role of the signaling in T-ALL we analyzed presence and expression profile of IKAROS splicing variants (using on-chip electrophoresis) in a cohort of 29 pediatric T-ALL patients. While we did not observe any link between NTAL or LAT levels and expression of different IKAROS isoforms, we found a close correlation between IKAROS isoforms IK1 and IK4a and another adaptors, PAG (major inhibitor of Src kinases in lymphocytes) and LIME (adaptor involved in signalization via CD4 and CD8). The two patients expressing IK1 and IK4A as dominant transcripts had >2 logs lower PAG (p=0.02) and LIME (p<0.1) expression levels compared to the rest of the cohort (n=27) showing IK2 as a dominant transcript. Based on this data, we propose that NTAL acts as a tumor suppressor enhancing proximal signaling of lymphocytes. Via phosphorylation of ERK, the NTAL sensitizes T-leukemic cells to corticosteroid induced apoptosis that is not mediated by FAS or FASL. The role of IKAROS isoforms and its connection with adaptor proteins is under investigation.The work was supported by grants MSM0021620813 and GAUK35607. DisclosuresNo relevant conflicts of interest to declare.

Overexpression of the Ikaros 6 isoform is restricted to t(4;11) acute lymphoblastic leukaemia in children and infants and has a role in B-cell survival.

The Ikaros transcription factor has been shown to play an important role in the differentiation of both myeloid and lymphoid lineages. Mice heterozygous for a dominant negative (DN) ikaros isoform develop T-cell leukaemia and lymphoma with 100% penetrance. Overexpression of DN Ikaros isoforms has been reported in some forms of leukaemia, such as childhood acute myelomonocytic and monocytic leukaemias, adult B-cell acute lymphoblastic leukaemias (B ALL) and in childhood and adult pre-B ALL. In this study, the expression of Ikaros isoforms in 49 infant and childhood leukaemia patients was analysed by reverse transcription polymerase chain reaction and Western blot analysis. We found overexpression of the DN Ikaros 6 (Ik6) isoform in a subset of leukaemia patients harbouring t(4;11) translocations. To further study the consequences of Ik6 overexpression in B ALL, we inducibly expressed Ik6 in BaF3 cells and found that Ik6 overexpression delayed cell death after interleukin-3 withdrawal, suggesting that overexpression of Ik6 found in t(4;11) B cells could contribute to leukaemogenesis by preventing the apoptosis of cells in an environment with reduced survival factors.

The Ikaros gene encodes a family of lymphocyte-restricted zinc finger DNA binding proteins, highly conserved in human and mouse.

The Ikaros gene is an essential regulator in the development and homeostasis of the mouse lymphopoietic system. To study the role of the Ikaros gene in the human lymphopoietic system, we cloned and characterized human Ikaros cDNAs. In the human, as in the mouse, differential splicing of Ikaros primary transcripts generates a family of lymphoid-restricted zinc finger DNA binding proteins, highly conserved in sequence composition and relative expression to the mouse homologues. Expression of Ikaros isoforms is highly restricted to the lymphopoietic system and is particularly enriched in maturing thymocytes. The Ikaros gene maps at a syntenic locus located on the short arm of human chromosome 7 and on mouse chromosome 11 next to the epidermal growth factor receptor (Egfr). The high degree of conservation of the Ikaros gene at the genetic and expression levels strongly suggests that it plays a fundamental role in the ontogeny of the lymphopoietic system across species.