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Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype.

Fahrenkrog B, Martinelli V, Nilles N, Fruhmann G, Chatel G, Juge S, Sauder U, Di Giacomo D, Mecucci C, Schwaller J - PLoS ONE (2016)

Bottom Line: Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML).In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors.Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Medicine, Université Libre de Bruxelles, Charleroi, Belgium.

ABSTRACT
Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

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Expression of Nup98 fusion proteins deregulates cell cycle progression.HeLa cells were transiently transfected with GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 and analyzed by live cell imaging and flow cytometry after release from a double-thymidine block. (A) Schematic presentation of the time course for live cell imaging and flow cytometric analysis. (B) GFP and differential interference contrast time-lapse images of HeLa cells after release from double-thymidine block are presented. Cells expressing Nup98-HOXA9 and Nup98-PMX1, respectively, show a delay in the re-entry into the cell cycle. Time is indicated in minutes. (C) Boxplot display of the quantitative analysis of the time between double-thymidine release and onset of mitosis. (D) Quantitative analysis of the relative time of mitosis onset. The differences between GFP, GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 expressing cells were statistically highly relevant (**P>0.01; ***P>0.001). (E) DNA flow cytometry of control, GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 expressing cells nine hours after release a double thymidine block. The differences between GFP, GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 expressing cells were statistically highly relevant (**P>0.01; ***P>0.001; ****P>0.0001).
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pone.0152321.g007: Expression of Nup98 fusion proteins deregulates cell cycle progression.HeLa cells were transiently transfected with GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 and analyzed by live cell imaging and flow cytometry after release from a double-thymidine block. (A) Schematic presentation of the time course for live cell imaging and flow cytometric analysis. (B) GFP and differential interference contrast time-lapse images of HeLa cells after release from double-thymidine block are presented. Cells expressing Nup98-HOXA9 and Nup98-PMX1, respectively, show a delay in the re-entry into the cell cycle. Time is indicated in minutes. (C) Boxplot display of the quantitative analysis of the time between double-thymidine release and onset of mitosis. (D) Quantitative analysis of the relative time of mitosis onset. The differences between GFP, GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 expressing cells were statistically highly relevant (**P>0.01; ***P>0.001). (E) DNA flow cytometry of control, GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 expressing cells nine hours after release a double thymidine block. The differences between GFP, GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 expressing cells were statistically highly relevant (**P>0.01; ***P>0.001; ****P>0.0001).

Mentions: LAP2α is known to regulate cell cycle progression and differentiation via the retinoblastoma-E2F pathway [40, 41]. Overexpression of LAP2α has been shown to reduce G1/S transition, whereas knock down of LAP2α enhanced cell cycle progression [40, 41]. We therefore asked whether Nup98 fusion expression might affect cell cycle progression. We carried out time lapse imaging of live cells expressing Nup98-HOXA9 and Nup98-PMX1, respectively, in combination with flow cytometric analyses (FACS) of the cell cycle. HeLa cells were transfected and subjected to a double thymidine block 8 hours after transfection (Fig 7A). A double thymidine block arrests cells at the G1/S border and cells will enter S phase after release into fresh medium and progress further in the cell cycle [42]. Live imaging was started 7–8 hours after release from double thymidine (Fig 7B, 430 minutes) and HeLa cells expressing GFP entered mitosis about 11 hours after the release (Fig 7B; top two rows, 670 minutes; Fig 6C) and progressed through mitosis within 45 minutes. HeLa cells expressing GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 showed a significant delayed entry into mitosis at only about 15 hours after the release from double thymidine block (Fig 7B; 880 and 850 minutes; Fig 7C). Progression through mitosis in both GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 expressing cells took about 45 minutes, similar to cells expressing GFP. Consistent with the time-lapse microscopy, FACS analyses of re-entry into the cycle revealed that Nup98-HOXA9 and Nup98-PMX1 expressing cells exhibited a delay in S phase onset. Double thymidine treatment arrested about 70% of the differently transfected HeLa cells in G1 (T0, Table 2 and S7A Fig). Nine hours after release from thymidine, 53% of GFP-expressing HeLa cells have reached G2, but only about 34% of Nup98-HOXA9 and about 23% of Nup98-PMX1 expressing cells (Fig 7E, Table 2), indicating a delay in G1/S transition. 13 hours after release it is not further possible to distinguish between cells that have passed mitosis and those delayed in cell cycle progression (T13, Table 2 and S7B Fig).


Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype.

Fahrenkrog B, Martinelli V, Nilles N, Fruhmann G, Chatel G, Juge S, Sauder U, Di Giacomo D, Mecucci C, Schwaller J - PLoS ONE (2016)

Expression of Nup98 fusion proteins deregulates cell cycle progression.HeLa cells were transiently transfected with GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 and analyzed by live cell imaging and flow cytometry after release from a double-thymidine block. (A) Schematic presentation of the time course for live cell imaging and flow cytometric analysis. (B) GFP and differential interference contrast time-lapse images of HeLa cells after release from double-thymidine block are presented. Cells expressing Nup98-HOXA9 and Nup98-PMX1, respectively, show a delay in the re-entry into the cell cycle. Time is indicated in minutes. (C) Boxplot display of the quantitative analysis of the time between double-thymidine release and onset of mitosis. (D) Quantitative analysis of the relative time of mitosis onset. The differences between GFP, GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 expressing cells were statistically highly relevant (**P>0.01; ***P>0.001). (E) DNA flow cytometry of control, GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 expressing cells nine hours after release a double thymidine block. The differences between GFP, GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 expressing cells were statistically highly relevant (**P>0.01; ***P>0.001; ****P>0.0001).
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Related In: Results  -  Collection

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pone.0152321.g007: Expression of Nup98 fusion proteins deregulates cell cycle progression.HeLa cells were transiently transfected with GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 and analyzed by live cell imaging and flow cytometry after release from a double-thymidine block. (A) Schematic presentation of the time course for live cell imaging and flow cytometric analysis. (B) GFP and differential interference contrast time-lapse images of HeLa cells after release from double-thymidine block are presented. Cells expressing Nup98-HOXA9 and Nup98-PMX1, respectively, show a delay in the re-entry into the cell cycle. Time is indicated in minutes. (C) Boxplot display of the quantitative analysis of the time between double-thymidine release and onset of mitosis. (D) Quantitative analysis of the relative time of mitosis onset. The differences between GFP, GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 expressing cells were statistically highly relevant (**P>0.01; ***P>0.001). (E) DNA flow cytometry of control, GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 expressing cells nine hours after release a double thymidine block. The differences between GFP, GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 expressing cells were statistically highly relevant (**P>0.01; ***P>0.001; ****P>0.0001).
Mentions: LAP2α is known to regulate cell cycle progression and differentiation via the retinoblastoma-E2F pathway [40, 41]. Overexpression of LAP2α has been shown to reduce G1/S transition, whereas knock down of LAP2α enhanced cell cycle progression [40, 41]. We therefore asked whether Nup98 fusion expression might affect cell cycle progression. We carried out time lapse imaging of live cells expressing Nup98-HOXA9 and Nup98-PMX1, respectively, in combination with flow cytometric analyses (FACS) of the cell cycle. HeLa cells were transfected and subjected to a double thymidine block 8 hours after transfection (Fig 7A). A double thymidine block arrests cells at the G1/S border and cells will enter S phase after release into fresh medium and progress further in the cell cycle [42]. Live imaging was started 7–8 hours after release from double thymidine (Fig 7B, 430 minutes) and HeLa cells expressing GFP entered mitosis about 11 hours after the release (Fig 7B; top two rows, 670 minutes; Fig 6C) and progressed through mitosis within 45 minutes. HeLa cells expressing GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 showed a significant delayed entry into mitosis at only about 15 hours after the release from double thymidine block (Fig 7B; 880 and 850 minutes; Fig 7C). Progression through mitosis in both GFP-Nup98-HOXA9 and GFP-Nup98-PMX1 expressing cells took about 45 minutes, similar to cells expressing GFP. Consistent with the time-lapse microscopy, FACS analyses of re-entry into the cycle revealed that Nup98-HOXA9 and Nup98-PMX1 expressing cells exhibited a delay in S phase onset. Double thymidine treatment arrested about 70% of the differently transfected HeLa cells in G1 (T0, Table 2 and S7A Fig). Nine hours after release from thymidine, 53% of GFP-expressing HeLa cells have reached G2, but only about 34% of Nup98-HOXA9 and about 23% of Nup98-PMX1 expressing cells (Fig 7E, Table 2), indicating a delay in G1/S transition. 13 hours after release it is not further possible to distinguish between cells that have passed mitosis and those delayed in cell cycle progression (T13, Table 2 and S7B Fig).

Bottom Line: Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML).In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors.Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Medicine, Université Libre de Bruxelles, Charleroi, Belgium.

ABSTRACT
Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

Show MeSH
Related in: MedlinePlus