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Nuclear export signal within CALM is necessary for CALM-AF10-induced leukemia.

Suzuki M, Yamagata K, Shino M, Aikawa Y, Akashi K, Watanabe T, Kitabayashi I - Cancer Sci. (2014)

Bottom Line: The CALM-AF10 fusion gene, which results from a t(10;11) translocation, is found in a variety of hematopoietic malignancies.Wild-type clathrin assembly lymphoid myeloid leukemia protein (CALM) primarily localizes in a diffuse pattern within the cytoplasm, whereas AF10 localizes in the nucleus; however, it is not clear where CALM-AF10 acts to induce leukemia.These results suggest that during leukemogenesis, CALM-AF10 plays its critical roles in the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematological Malignancy, National Cancer Center Research Institute, Tokyo, Japan.

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The nuclear export signal within clathrin assembly lymphoid myeloid leukemia protein (CALM) is critical for in vitro immortalization of cells by CALM-AF10. (a) Serial colony-replating assays of murine bone-marrow cells transduced with FLAG-tagged wild-type and mutant CALM-AF10. In each round of replating, 3 × 104 transduced bone-marrow cells were plated. Bars represent the numbers of colonies. (b) Hoxa cluster and Meis1 expression in cells transduced with wild-type or mutant CALM-AF10. RNA transcripts were analyzed by real-time PCR of murine bone-marrow cells transduced with wild-type and mutant CALM-AF10 in vitro. Expression levels of Hoxa5, Hoxa7, Hoxa9, Hoxa10 and Meis1 were normalized against Actb expression and compared with the levels in vector-transfected whole bone-marrow cells. Data are shown as means ± SEM from three independent samples. *P < 0.05; **P < 0.01. (vs normal bone-marrow cells). CA WT, wild-type CALM-AF10.
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fig02: The nuclear export signal within clathrin assembly lymphoid myeloid leukemia protein (CALM) is critical for in vitro immortalization of cells by CALM-AF10. (a) Serial colony-replating assays of murine bone-marrow cells transduced with FLAG-tagged wild-type and mutant CALM-AF10. In each round of replating, 3 × 104 transduced bone-marrow cells were plated. Bars represent the numbers of colonies. (b) Hoxa cluster and Meis1 expression in cells transduced with wild-type or mutant CALM-AF10. RNA transcripts were analyzed by real-time PCR of murine bone-marrow cells transduced with wild-type and mutant CALM-AF10 in vitro. Expression levels of Hoxa5, Hoxa7, Hoxa9, Hoxa10 and Meis1 were normalized against Actb expression and compared with the levels in vector-transfected whole bone-marrow cells. Data are shown as means ± SEM from three independent samples. *P < 0.05; **P < 0.01. (vs normal bone-marrow cells). CA WT, wild-type CALM-AF10.

Mentions: We next investigated whether the NES within CALM-AF10 is required for leukemogenesis. To this end, primary murine bone-marrow stem/progenitor cells (HSPC) were infected with retrovirus encoding CALM-AF10, CALMNES4A-AF10, NES2-AF10 and mAF10. Serial-replating assays revealed that both CALM-AF10 and NES2-AF10 immortalized HSPC, and that the cells formed colonies for at least five rounds of replating (Fig.2a). By contrast, neither mAF10 nor CALMNES4A-AF10, which lacks a functional CALM NES, could immortalize cells. Transduced cells with elevated colony-forming abilities also exhibited upregulation of the Hoxa cluster (Hoxa5, Hoxa7, Hoxa9 and Hoxa10) and Meis1 genes (Fig.2b)7,9. These results indicated that the CALM NES is necessary for CALM-AF10 to immortalize hematopoietic stem/progenitor cells.


Nuclear export signal within CALM is necessary for CALM-AF10-induced leukemia.

Suzuki M, Yamagata K, Shino M, Aikawa Y, Akashi K, Watanabe T, Kitabayashi I - Cancer Sci. (2014)

The nuclear export signal within clathrin assembly lymphoid myeloid leukemia protein (CALM) is critical for in vitro immortalization of cells by CALM-AF10. (a) Serial colony-replating assays of murine bone-marrow cells transduced with FLAG-tagged wild-type and mutant CALM-AF10. In each round of replating, 3 × 104 transduced bone-marrow cells were plated. Bars represent the numbers of colonies. (b) Hoxa cluster and Meis1 expression in cells transduced with wild-type or mutant CALM-AF10. RNA transcripts were analyzed by real-time PCR of murine bone-marrow cells transduced with wild-type and mutant CALM-AF10 in vitro. Expression levels of Hoxa5, Hoxa7, Hoxa9, Hoxa10 and Meis1 were normalized against Actb expression and compared with the levels in vector-transfected whole bone-marrow cells. Data are shown as means ± SEM from three independent samples. *P < 0.05; **P < 0.01. (vs normal bone-marrow cells). CA WT, wild-type CALM-AF10.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4317939&req=5

fig02: The nuclear export signal within clathrin assembly lymphoid myeloid leukemia protein (CALM) is critical for in vitro immortalization of cells by CALM-AF10. (a) Serial colony-replating assays of murine bone-marrow cells transduced with FLAG-tagged wild-type and mutant CALM-AF10. In each round of replating, 3 × 104 transduced bone-marrow cells were plated. Bars represent the numbers of colonies. (b) Hoxa cluster and Meis1 expression in cells transduced with wild-type or mutant CALM-AF10. RNA transcripts were analyzed by real-time PCR of murine bone-marrow cells transduced with wild-type and mutant CALM-AF10 in vitro. Expression levels of Hoxa5, Hoxa7, Hoxa9, Hoxa10 and Meis1 were normalized against Actb expression and compared with the levels in vector-transfected whole bone-marrow cells. Data are shown as means ± SEM from three independent samples. *P < 0.05; **P < 0.01. (vs normal bone-marrow cells). CA WT, wild-type CALM-AF10.
Mentions: We next investigated whether the NES within CALM-AF10 is required for leukemogenesis. To this end, primary murine bone-marrow stem/progenitor cells (HSPC) were infected with retrovirus encoding CALM-AF10, CALMNES4A-AF10, NES2-AF10 and mAF10. Serial-replating assays revealed that both CALM-AF10 and NES2-AF10 immortalized HSPC, and that the cells formed colonies for at least five rounds of replating (Fig.2a). By contrast, neither mAF10 nor CALMNES4A-AF10, which lacks a functional CALM NES, could immortalize cells. Transduced cells with elevated colony-forming abilities also exhibited upregulation of the Hoxa cluster (Hoxa5, Hoxa7, Hoxa9 and Hoxa10) and Meis1 genes (Fig.2b)7,9. These results indicated that the CALM NES is necessary for CALM-AF10 to immortalize hematopoietic stem/progenitor cells.

Bottom Line: The CALM-AF10 fusion gene, which results from a t(10;11) translocation, is found in a variety of hematopoietic malignancies.Wild-type clathrin assembly lymphoid myeloid leukemia protein (CALM) primarily localizes in a diffuse pattern within the cytoplasm, whereas AF10 localizes in the nucleus; however, it is not clear where CALM-AF10 acts to induce leukemia.These results suggest that during leukemogenesis, CALM-AF10 plays its critical roles in the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematological Malignancy, National Cancer Center Research Institute, Tokyo, Japan.

Show MeSH
Related in: MedlinePlus