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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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Characterization of leukemic cells in vivo. (a) Flow cytometric analysis of leukemic cells. Murine bone-marrow cells were prepared from mice that developed leukemia after receiving transplantation of tumor cells transduced with CALM-AF10 or NES2-AF10, and were co-stained for Gr-1, Mac-1, colony stimulating factor 1 receptor (CSF1R) and c-kit; data are representative of CALM-AF10 primary transplantation (n = 3) and NES2-AF10 primary transplantation (n = 3). (b) Hoxa cluster and Meis1 expression in mice receiving cells transduced with wild-type and mutant CALM-AF10. RNA transcripts were analyzed by real-time PCR of bone-marrow cells in mice that developed leukemia after CALM-AF10 and NES2-AF10 bone-marrow transplantation. Expression levels of Hoxa5, Hoxa7, Hoxa9, Hoxa10 and Meis1 were normalized against Actb and compared with wild-type whole bone marrow. Data are shown as means ± SEM from three independent leukemic mice. *P < 0.05; **P < 0.01 (vs normal bone-marrow cells).
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fig04: Characterization of leukemic cells in vivo. (a) Flow cytometric analysis of leukemic cells. Murine bone-marrow cells were prepared from mice that developed leukemia after receiving transplantation of tumor cells transduced with CALM-AF10 or NES2-AF10, and were co-stained for Gr-1, Mac-1, colony stimulating factor 1 receptor (CSF1R) and c-kit; data are representative of CALM-AF10 primary transplantation (n = 3) and NES2-AF10 primary transplantation (n = 3). (b) Hoxa cluster and Meis1 expression in mice receiving cells transduced with wild-type and mutant CALM-AF10. RNA transcripts were analyzed by real-time PCR of bone-marrow cells in mice that developed leukemia after CALM-AF10 and NES2-AF10 bone-marrow transplantation. Expression levels of Hoxa5, Hoxa7, Hoxa9, Hoxa10 and Meis1 were normalized against Actb and compared with wild-type whole bone marrow. Data are shown as means ± SEM from three independent leukemic mice. *P < 0.05; **P < 0.01 (vs normal bone-marrow cells).

Mentions: To determine whether CALM-AF10 and NES2-AF10 can induce leukemia in mice, we injected bone-marrow progenitor cells transduced with CALM-AF10 and NES2-AF10 into lethally irradiated mice. Seven out of eight mice transplanted with cells expressing CALM-AF10 developed leukemia within 6 months after transplantation (Fig.3a), and all mice transplanted with cells expressing NES2-AF10 developed leukemia within 3 months after transplantation. When cells prepared from bone marrow of these leukemic mice were transplanted into secondary recipient mice, all recipients promptly developed leukemia (medians: CALM-AF10 donors, 21 days [n = 4]; NES2-AF10 donors, 25 days [n = 9]). Morphological analysis revealed large populations of blast cells in leukemic mice receiving cells transduced with either CALM-AF10 or NES2-AF10 (Fig.3b,c). Flow cytometry analysis showed that cells expressing CALM-AF10 and NES2-AF10 in the bone marrow cells of primary recipient mice were Mac1+, CSF1R+ and c-kit+ (Fig.4a). Moreover, as shown in Figure4b, Hoxa5, Hoxa7, Hoxa9, Hoxa10 and Meis1 expression levels were upregulated in cells expressing CALM-AF10 and NES2-AF10 compared with normal bone marrow cells, although upregulation of Hoxa5 and Meis1 in primary recipient mice harboring NES2-AF10 was not significant (P = 0.084 and P = 0.093, respectively). These data demonstrate that the NES within CALM-AF10 is a critical element for induction of leukemia.


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)

Characterization of leukemic cells in vivo. (a) Flow cytometric analysis of leukemic cells. Murine bone-marrow cells were prepared from mice that developed leukemia after receiving transplantation of tumor cells transduced with CALM-AF10 or NES2-AF10, and were co-stained for Gr-1, Mac-1, colony stimulating factor 1 receptor (CSF1R) and c-kit; data are representative of CALM-AF10 primary transplantation (n = 3) and NES2-AF10 primary transplantation (n = 3). (b) Hoxa cluster and Meis1 expression in mice receiving cells transduced with wild-type and mutant CALM-AF10. RNA transcripts were analyzed by real-time PCR of bone-marrow cells in mice that developed leukemia after CALM-AF10 and NES2-AF10 bone-marrow transplantation. Expression levels of Hoxa5, Hoxa7, Hoxa9, Hoxa10 and Meis1 were normalized against Actb and compared with wild-type whole bone marrow. Data are shown as means ± SEM from three independent leukemic mice. *P < 0.05; **P < 0.01 (vs normal bone-marrow cells).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig04: Characterization of leukemic cells in vivo. (a) Flow cytometric analysis of leukemic cells. Murine bone-marrow cells were prepared from mice that developed leukemia after receiving transplantation of tumor cells transduced with CALM-AF10 or NES2-AF10, and were co-stained for Gr-1, Mac-1, colony stimulating factor 1 receptor (CSF1R) and c-kit; data are representative of CALM-AF10 primary transplantation (n = 3) and NES2-AF10 primary transplantation (n = 3). (b) Hoxa cluster and Meis1 expression in mice receiving cells transduced with wild-type and mutant CALM-AF10. RNA transcripts were analyzed by real-time PCR of bone-marrow cells in mice that developed leukemia after CALM-AF10 and NES2-AF10 bone-marrow transplantation. Expression levels of Hoxa5, Hoxa7, Hoxa9, Hoxa10 and Meis1 were normalized against Actb and compared with wild-type whole bone marrow. Data are shown as means ± SEM from three independent leukemic mice. *P < 0.05; **P < 0.01 (vs normal bone-marrow cells).
Mentions: To determine whether CALM-AF10 and NES2-AF10 can induce leukemia in mice, we injected bone-marrow progenitor cells transduced with CALM-AF10 and NES2-AF10 into lethally irradiated mice. Seven out of eight mice transplanted with cells expressing CALM-AF10 developed leukemia within 6 months after transplantation (Fig.3a), and all mice transplanted with cells expressing NES2-AF10 developed leukemia within 3 months after transplantation. When cells prepared from bone marrow of these leukemic mice were transplanted into secondary recipient mice, all recipients promptly developed leukemia (medians: CALM-AF10 donors, 21 days [n = 4]; NES2-AF10 donors, 25 days [n = 9]). Morphological analysis revealed large populations of blast cells in leukemic mice receiving cells transduced with either CALM-AF10 or NES2-AF10 (Fig.3b,c). Flow cytometry analysis showed that cells expressing CALM-AF10 and NES2-AF10 in the bone marrow cells of primary recipient mice were Mac1+, CSF1R+ and c-kit+ (Fig.4a). Moreover, as shown in Figure4b, Hoxa5, Hoxa7, Hoxa9, Hoxa10 and Meis1 expression levels were upregulated in cells expressing CALM-AF10 and NES2-AF10 compared with normal bone marrow cells, although upregulation of Hoxa5 and Meis1 in primary recipient mice harboring NES2-AF10 was not significant (P = 0.084 and P = 0.093, respectively). These data demonstrate that the NES within CALM-AF10 is a critical element for induction of leukemia.

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