Cytokinesis failure occurs in Fanconi anemia pathway-deficient murine and human bone marrow hematopoietic cells

Abstract

Fanconi anemia (FA) is a genomic instability disorder characterized by bone marrow failure and cancer predisposition. FA is caused by mutations in any one of several genes that encode proteins cooperating in a repair pathway and is required for cellular resistance to DNA crosslinking agents. Recent studies suggest that the FA pathway may also play a role in mitosis, since FANCD2 and FANCI, the 2 key FA proteins, are localized to the extremities of ultrafine DNA bridges (UFBs), which link sister chromatids during cell division. However, whether FA proteins regulate cell division remains unclear. Here we have shown that FA pathway-deficient cells display an increased number of UFBs compared with FA pathway-proficient cells. The UFBs were coated by BLM (the RecQ helicase mutated in Bloom syndrome) in early mitosis. In contrast, the FA protein FANCM was recruited to the UFBs at a later stage. The increased number of bridges in FA pathway-deficient cells correlated with a higher rate of cytokinesis failure resulting in binucleated cells. Binucleated cells were also detectable in primary murine FA pathway-deficient hematopoietic stem cells (HSCs) and bone marrow stromal cells from human patients with FA. Based on these observations, we suggest that cytokinesis failure followed by apoptosis may contribute to bone marrow failure in patients with FA.

Figure 1. FA pathway–deficient cells exhibit more PICH-BLM bridges in mitosis than do cDNA-complemented or WT cells.

(A) FA-G cells transduced with empty vector (FA-G+V) or reconstituted with FANCG cDNA (FA-G+G) were stained for PICH (red), BLM (green), and DNA (Hoechst; blue). A representative FA-G plus vector mitotic cell with multiple PICH-BLM bridges is shown in anaphase-telophase and in late telophase. Original magnification, ×63. (B) Frequency of mitoses displaying PICH-BLM bridges in FANCG-deficient and -proficient cells. Cells were scored for the presence of PICH-BLM bridges in anaphase-telophase or in late telophase. Data are mean and SD from 2 independent experiments. (C) Distribution of FA-G cells plus vector or FANCG cDNA based on the number of PICH-BLM bridges per mitotic cell. (D) Frequency of mitoses displaying BLM bridges in vector-transduced and reconstituted FA-A cells (FA-A+V and FA-A+A, respectively). Data are mean and SEM from 3 independent experiments. (E) Frequency of mitoses displaying BLM bridges in shScramble, shFANCA, or shFANCI cells. Data are mean and SD from 2 independent experiments. Numbers within bars denote number of mitotic cells analyzed. **P < 0.02, ***P < 0.01, Student’s t test.

Figure 2. Localization of FANCM protein to PICH-BLM bridges.

(A) Representative images for BLM and FANCM bridges in HeLa cells in anaphase and late telophase. Cells were stained for DNA (blue), microtubules (red), and BLM or FANCM (green). Original magnification, ×100. (B) Frequency of mitotic HeLa cells displaying BLM or FANCM bridges in anaphase and late telophase. Data are mean and SD of 2 independent experiments. (C) Frequency of HeLa cells displaying FANCM bridges in late telophase after treatment with control siRNA (siLacZ) or BLM siRNA for 72 hours. Data are mean and SEM of 3 independent experiments. (D) Representative confocal images of FANCM bridges (green) linking DNA fragile sites marked by FANCD2 foci (red color and arrows). Insets show higher-magnification views of FANCM bridges connecting FANCD2 foci. HeLa cells were treated for 18 hours with aphidicolin (0.3 μM) and then released for 6 hours before fixation. Original magnification, ×100; ×250 (insets). (E) Frequency of shScramble, shFANCA, and shFANCI cells displaying FANCM bridges in late telophase. Data are mean and SD from 2 independent experiments. (F) Frequency of control cells (shCtrl), cells stably knocked down for FANCM (shM), or FANCM-deficient cells complemented with WT cDNA (shM+M WT) or expressing FANCMK117R displaying BLM bridges (shM+M K117R) in mitosis. Data are mean and SEM from 3 independent experiments. Numbers within bars denote number of mitotic cells analyzed. *P < 0.05, **P < 0.02, ***P < 0.01, Student’s t test.

Figure 3. Disruption of the FA pathway results in cytokinesis failure and an increase in multinucleated cells.

(A) FA-A and FA-G fibroblasts and corrected counterparts, as well as shScramble, shFANCA, and shFANCI cells, were stained for microtubules and DNA. Mononucleated as well as bi- and multinucleated cells were scored, and the frequency of multinucleated cells (i.e., bi- and multi-) for each type was calculated. (B) Frequency of cytokinesis failure in shScramble and shFANCA cells, followed for 24 hours by live cell imaging. (C) Still frames of representative successful and failed cytokinesis; see Supplemental Movies 1 and 2, respectively. Time is indicated in hours and minutes. Scale bars: 10 μM. (D) HeLa cells transiently knocked down for a panel of Fanconi genes as well as BLM were analyzed for the presence of bi- and multinucleated cells. Data are mean and SEM from 3 independent experiments (every siRNA was independently compared with siLacZ). (E) Control cells and cells stably knocked down for FANCM, complemented with the WT FANCM cDNA, and expressing FANCMK117R were analyzed for the presence of bi- and multinucleated cells. Numbers within bars denote number of mitotic cells analyzed. ***P < 0.01, Student’s t test.

Figure 4. Increased cytokinesis failure in murine Fancg^–/– and Fancd2^–/– HSCs and in human FA bone marrow stromal cells.

(A) LSK cells from Fancg^–/– and Fancd2^–/– mice and their respective WT siblings were grown for 48 hours before staining for microtubules (red), nuclear membrane (Lap2; green), and DNA (blue). Representative images of mononucleated as well as binucleated Fancd2^–/– LSK cells are shown (note the Lap2 bridge between the 2 nuclei in the third column). Original magnification, ×63. (B) Frequency of binucleated cells in LSK population from bone marrow of 4 pairs of Fancg^–/– and WT siblings and 3 pairs of Fancd2^–/– and WT siblings. At right, the fold increase in binucleated cells between FA pathway–deficient and WT samples was calculated for each pair of mice; data are mean and SEM. (C) Frequency of binucleated cells in the bone marrow stromal cell population of 3 healthy donors (control, mean and SEM shown) and 3 FA patients. (D) LSK cells from 3 pairs each of Fancd2^–/– and WT and Fancg^–/– and WT siblings were grown for 48 hours before staining with annexin V. The absolute frequencies of annexin V–positive apoptotic LSK cells and the fold increase in the number of apoptotic cells is shown. Numbers within and above bars denote number of cells analyzed. **P < 0.02, *P < 0.05, Student’s t test.