Figure 1.
Ikaros 11 is a novel, non-canonical splice variant of the Ikaros gene.
(A) Diagrammatic representation of Ikaros isoforms 1–10 with their functional domains. (B) Schematic representation of the novel splice variant Ik11. (DBD, DNA Binding Domain; AD, Activation Domain; DD, Dimerization Domain, F1–F6, Zinc Finger modules). (C) Amino acid sequence alignment of the full-length Ik1 and Ik11. Exon positions are indicated. Exon 3B has been previously described [16], [25], [36] but is currently not identified as an exon in Gene Bank.
Figure 2.
Ik11 expression is restricted to lymph nodes and peripheral lymphocytes.
(A) Real-time PCR analysis of Ikaros mRNA in human cDNAs from normal thymus, spleen, lymph nodes and bone marrow. The Ik11 levels are expressed as fold change relative to expression in thymus and normalized to the expression of GAPDH. Each bar represents the average ± SD of three replicates. (B) Real-time PCR analysis of Ik11 transcripts in magnetic bead-purified human leukocyte subsets (CD4+ and CD8+ T cells, CD19+ B cells and CD14+ monocytes). The Ik11 levels are expressed as fold change relative to expression in PBLs obtained from the healthy donor #1 (see Figure S3) and normalized to the expression of GAPDH. Each bar represents the average ± SD of three replicates. (C) Sequencing of Ik11 real-time PCR products from lymph nodes. The electropherogram shows the sequence corresponding to the junction fragments half exon 5/half exon 8.
Figure 3.
Ik11 acts as a dominant negative isoform.
(A) In vitro co-immunoprecipitation of Ik2 with the short isoforms Ik11 or Ik6. Ik2-myc, biotinylated-Ik11 and biotinylated-Ik6 were generated by in vitro transcription/translation. After 1 h incubation of Ik2-Myc with Ik11 or Ik6, the Ik2 complexes were immunoprecipitated with an anti-Myc antibody and subjected to Western blot analysis as indicated (lanes 1 and 2). Lanes 3, 4 and 5 contained the three in vitro translated proteins without immunoprecipitation. (B) In vivo heterodimerization of Ik2 with the splice variants Ik11 or Ik6. The 293T HEK cell line was co-transfected with pcDNA3.1/Myc-HysB-Ik2 along with pcDNA3.1-Ik6 or pcDNA3.1-Ik11. The Ik2 immunoprecipitation was performed with an anti-Myc antibody and the immune-complexes were analyzed by Western blotting as indicated. (C) Luciferase assay showing functional dominant-negative activity of Ik11. The 293T HEK cell line was co-transfected with pcDNA3.1-Ik2, pcDNA3.1-Ik6 or pcDNA3.1-Ik11 or their combinations along with a reporter-LUC construct driven by a promoter containing Ikaros-binding sites. Mean ± SD of triplicate wells is shown (***p<0.001). Data shown are representative of three different experiments.
Figure 4.
Ik2 subcellular localization changes in presence of Ik11.
(A) Subcellular localization of Ik2 (panels a–d), Ik11 (panels e-g) and Ik6 (panels h–j). The Cos7 cell line was transfected with pcDNA3.1/Myc-HysB-Ik2, pcDNA3.1-Ik6 or pcDNA3.1-Ik11 constructs and the cellular localization of each isoform was analyzed by confocal microscopy. Immunofluorescence localization of Ik2 was assessed by anti-Myc antibody and Texas Red dye conjugated AffiniPure Goat anti-mouse IgG (H+L) (red fluorescence); Ik6 and Ik11 were detected with anti-Ikaros antibody and Fluorescein (FITC)-conjugated AffiniPure Goat anti-Rabbit IgG (H+L) (green fluorescence). Nuclei were stained with Hoechst 33258 (panels a, e, h, blue fluorescence). Merged images of double fluorescence (Hoechst localization of nuclei plus Ikaros staining) are shown for all of the three isoforms (Ik2: panels c and d, scale bar equals to 50 and 10 microns respectively; Ik11: panel g; Ik6: panel j). x40 magnification (panels a–c, e–j). Panel d was an x2.3 zoom of the white box field indicated in panel c. (B) Graphic representation of Ik2, Ik11 and Ik6 subcellular localization. The analysis was conducted counting nuclear or cytoplasmic staining, or both (nuclear+cytoplasmic), of the three isoforms as percent point. 5 fields were counted for each transfection. (C) Confocal triple immunofluorescence images of Hoechst 33258 plus Ik2-myc and Ik11 or Ik6. Cos7 cells were co-transfected with either pcDNA3.1/Myc-HysB-Ik2 and pcDNA3.1-Ik11 (a–e) or pcDNA3.1/Myc-HysB-Ik2 and pcDNA3.1-Ik6 (f–i) expression vectors. Staining for Ik11 (green fluorescence), Ik6 (green fluorescence), Ik2 (red fluorescence) and Hoechst 33258 (blue fluorescence) were performed as described in (A). Merged images of triple fluorescence (Hoechst localization of nuclei plus co-localization of Ik2/Ik11 or Ik2/Ik6) were illustrated in panels d-e and panel j, respectively. Scale bar were equals to 50 microns (panels a-d and panels f–j, x40 objective) and 10 microns (panel e, x6 zoom of the white box field indicated in panel d). (D) Graphic representation of Ik2 subcellular localization when it is transfected alone or in combination with Ik11 and Ik6. The analysis was conducted counting Ik2 nuclear or cytoplasmic staining, or both (nuclear+cytoplasmic), as percent point. 5 fields were counted for each transfection.
Figure 5.
Ik11 overexpression promotes cell proliferation.
(A–B) Analysis of cell proliferation in Raw 264 (A) and BJAB (B) cell lines. Cells were transfected with pcDNA3.1-Ik2, pcDNA3.1-Ik6, pcDNA3.1-Ik11 or empty vector. Proliferation was determined by the MTS assay at 24 h (BJAB) or 48 h (RAW 264) and calculated as the percent of control (pcDNA3.1). Mean ± SD is shown (*p<0.05; **p<0.001). Data shown are representative of three different experiments. (C–E) Western blot analysis of p27, p21 and cyclin E in RAW 264 (C), BJAB (D) and K562 (E) cell lines.
Figure 6.
Ik11 overexpression protects against apoptosis.
Evaluation of apoptosis by measurement of caspase-3 activity (A), examination of PARP cleavage products (B) and determination of mono- and oligo-nucleosome enrichment (C). Raw 264 cells were transfected with pcDNA3.1-Ik2, pcDNA3.1-Ik6, pcDNA3.1-Ik11 or empty vector and were incubated with 100 nM staurosporine or vehicle for 14 hours. Empty vector-transfected cells were also treated with 50 µM Z-VAD-FMK, a general caspase inhibitor. Values were calculated as percent of control (pcDNA3.1). Mean ± SD is shown (***p<0.0001) (A,C). Data shown are representative of three different experiments. (D) Western blot analysis of Bax protein. Raw 264 cells were transfected with Ikaros isoforms and treated with staurosporine as previously described. Protein levels of Bax and Bax/p18 cleavage products are shown.
Figure 7.
Increased Ik11 expression in several hematological malignancies.
(A, B, C) Real-time PCR analysis of Ik11 and Ik6 mRNAs in samples of chronic myeloid leukemia (A, n = 21), acute lymphoblastic leukemia (B, n = 11) and myelodysplastic syndromes (C, n = 7). The Ik11 levels are expressed as fold change relative to expression in PBLs obtained from the healthy donor #1 (see Figure S3) and normalized to the expression of GAPDH. Each bar represents the average ± SD of three replicates. (D) Expression of Ik11 and Ik6 in commercial samples of lymphoproliferative disorders (n = 3). 1 = Poorly differentiated malignant lymphoma; 2 = Hodgkin’s lymphoma; 3 = non-Hodgkin’s lymphoma, diffuse; + = PCR positive controls. (E, F) Real-time PCR analysis of Ik11 and Ik6 mRNAs in samples of lymphoma (E, n = 7) and chronic lymphoblastic leukemia (F, n = 22). The Ik11 levels are expressed as fold change relative to expression in PBLs obtained from the healthy donor #1 (see Figure S3) and normalized to the expression of GAPDH. Each bar represents the average ± SD of three replicates.
Table 1.
Clinical data in patients with Lymphoma.
Table 2.
Clinical data in patients with Chronic Lymphocytic Leukemia.