The ARF tumor suppressor prevents chromosomal instability and ensures mitotic checkpoint fidelity through regulation of Aurora B

Abstract

The ARF tumor suppressor is part of the CDKN2A locus and is mutated or undetectable in numerous cancers. The best-characterized role for ARF is in stabilizing p53 in response to cellular stress. However, ARF has tumor suppressive functions outside this pathway that have not been fully defined. Primary mouse embryonic fibroblasts (MEFs) lacking the ARF tumor suppressor contain abnormal numbers of chromosomes. However, no role for ARF in cell division has previously been proposed. Here we demonstrate a novel, p53-independent role for ARF in the mitotic checkpoint. Consistent with this, loss of ARF results in aneuploidy in vitro and in vivo. ARF(-/-) MEFs exhibit mitotic defects including misaligned and lagging chromosomes, multipolar spindles, and increased tetraploidy. ARF(-/-) cells exhibit overexpression of Mad2, BubR1, and Aurora B, but only overexpression of Aurora B phenocopies mitotic defects observed in ARF(-/-) MEFs. Restoring Aurora B to near-normal levels rescues mitotic phenotypes in cells lacking ARF. Our results define an unexpected role for ARF in chromosome segregation and mitotic checkpoint function. They further establish maintenance of chromosomal stability as one of the additional tumor-suppressive functions of ARF and offer a molecular explanation for the common up-regulation of Aurora B in human cancers.

FIGURE 1:

ARF loss causes aneuploidy in vitro. (A) Diploid MEF chromosome spread with 40 chromosomes. Scale bar, 10 μm. (B) Average aneuploidy in MEFs. n = 3 experiments of 50 spreads each. (C) Histogram of chromosome spreads from B. Inset, enlarged histogram showing the percentages of near-tetraploid MEFs. (D) Tetraploid MEF chromosome spread with 80 chromosomes. Scale bar, 10 μm. (E) Average tetraploidy in MEFs. n = 3 experiments of 100 spreads each. (F) Binucleate MEF. Red, F-actin stained with phalloidin. Blue, DNA. Scale bar, 20 μm. (G) Average percentage of binucleation in MEFs of the indicated genotypes. n = 250 cells from each of three independent experiments. **p < 0.001.

FIGURE 2:

ARF is required to maintain chromosomal stability in vivo. (A) Chromosome spread from a mouse splenocyte with 40 chromosomes. Scale bar, 10 μm. (B) Average aneuploidy in 5-mo-old mouse splenocytes. n = 3 independent experiments of 50 spreads each. (C) Histogram of chromosome spreads from B. No tetraploid spreads were observed in splenocytes. Mitotic indices of splenocytes were comparable in wild-type (0.52 ± 0.18%) and ARF-null (0.62 ± 0.03%) animals. n ≥ 930 cells from three animals. (D) Murine intestine labeled with FISH probe to chromosome 11. Top right is labeled to indicate nuclear boundaries and number of chromosome 11 signals in each cell. (E) Percentage of cells of the indicated genotypes with greater or less than two copies of chromosome 11. (F) Percentage of cells with the indicated number of copies of chromosome 11. *p < 0.05; **p < 0.001.

FIGURE 3:

ARF^−/− cells exhibit mitotic defects. (A, B) ARF^−/− MEFs have increased levels of misaligned chromosomes. (A) Left, normal metaphase. Right, cell with metaphase plate and misaligned chromosome (arrow). (B) Percentage of MEFs of the indicated genotypes containing visible metaphase plates and misaligned chromosomes. n > 50 metaphases from three independent experiments. (C, D) ARF loss causes congression defects. (C) Left, image of MG132-treated cell with fully congressed chromosomes. Right, image of MG132-treated cell with incompletely aligned chromosomes. (D) Quantitation of the percentage of wild-type and ARF^−/− cells that successfully aligned their chromosomes after 3 h treatment with 10 μM MG132 (left) or after 18 h of 100 μM monastrol to induce monopolar spindles, followed by washout into 3 h of MG132 (mon→MG; right). (E, F) MEFs lacking ARF have increased levels of lagging chromosomes in anaphase and telophase. (E) Left, normal anaphase. Right, abnormal anaphase cell with lagging chromosome indicated by arrow. (F) Percentage of cells of the indicated genotype with lagging chromosomes. n >100 anaphases and telophases from thee independent experiments. (G, H) ARF^−/− MEFs have an elevated frequency of supernumery centrosomes. (G) Interphase cells with two (left) or four centrosomes (right), denoted by arrows. (H) Percentage of interphase cells with abnormal numbers of centrosomes. n > 250 cells from each of three independent experiments. (I–K) ARF loss results in multipolar spindles. (I) Example of a multipolar spindle in an ARF^−/− MEF. (J) Percentage of MEFs with abnormal spindles. (K) Histogram showing number of poles per spindle. n > 100 mitotic cells from each of three independent experiments. Scale bars, 5 μm. *p < 0.05.

FIGURE 4:

ARF^−/− cells have a weakened mitotic checkpoint. (A) Mitotic index of MEFs treated with colcemid for the specified number of hours. n = 250 cells at each time point from each of three independent experiments. (B) Duration of mitosis in MEFs treated with colcemid, as assessed by time-lapse microscopy. n = 100 cells from three independent experiments. (C) ARF transfection rescues mitotic checkpoint activity in ARF^−/− MEFs. After 32 h of transfection with ARF-YFP or empty vector, MEFs were treated with colcemid or vehicle for 16 h before analysis of mitotic index. n = 250 cells from each of three independent experiments. (D) Mitotic index of MEFs infected with retroviruses expressing empty vector or wild-type or V24E p14ARF. Green fluorescent protein (GFP) was also expressed from an internal ribosomal entry site. After 32 h of infection, cells were treated with colcemid for 16 h before analysis of mitotic index. n > 250 cells from each of three independent experiments. (E) Percentage of metaphase cells with misaligned chromosomes after 72 h of infection with retroviruses expressing empty vector or wild-type or V24E p14ARF. Both wild-type p14ARF and the MDM2-binding-domain mutant V24E rescue the occurrence of misaligned chromosomes in ARF^−/− MEFs. n > 50 metaphases from three independent experiments. (F) Percentage of anaphase or telophase MEFs with lagging chromosomes after infection with empty vector or wild-type or V24E p14ARF. Both wild-type and V24E p14ARF reduce the incidence of lagging chromosomes in ARF^−/− cells. n > 100 anaphases and telophases from three independent experiments. (G) Levels of the mitotic checkpoint components Mad2 and BubR1 are elevated in ARF^−/− MEFs, whereas levels of Bub1 and CENP-E remain unchanged. Tubulin, loading control. *p < 0.05.

FIGURE 5:

Mitotic functions of ARF are p53-independent. (A) Percentage of MEFs with misaligned chromosomes in metaphase. Loss of ARF increases the incidence of misaligned chromosomes in p53^−/− cells. n > 80 metaphases from three independent experiments. (B) Percentage of MEFs with lagging chromosomes in anaphase or telophase. Although loss of either ARF or p53 increases the percentage of cells containing lagging chromosomes, reduction of ARF in p53^−/− cells causes a further increase, consistent with a p53-independent role for ARF in preventing chromosome lagging. n > 100 anaphases and telophases from each of three independent experiments. (C) Mitotic index of MEFs of the indicated genotypes ± 16 h of colcemid treatment. ARF, but not p53, is required for the mitotic checkpoint–mediated increase in mitotic index in response to colcemid. n > 250 cells from each of three independent experiments. *p < 0.05.

FIGURE 6:

Aurora B expression is elevated in ARF^−/− cells in vitro and in vivo. (A) Immunoblot showing increased levels of Aurora B in MEFs with reduced expression of ARF. (B) Splenocytes from ARF^−/− mice express heightened amounts of Aurora B relative to splenocytes from wild-type animals. Coomassie is used as a loading control.

FIGURE 7:

Elevated levels of Aurora B in ARF^−/− cells are due to increased protein stability. (A) Quantitative real-time PCR examining relative Aurora B mRNA levels in ARF^+/+ and ARF^−/− cells. Two different primer sets show a modest, nonsignificant increase in Aurora B transcript levels in ARF^−/− primary MEFs. p = 0.285 and 0.1088 for primer sets 1 and 2, respectively, as assessed by Wilcoxon signed rank test. n = 3. (B) Immunoblots showing Aurora B levels after the indicated number of hours of cyclohexamide treatment to prevent new protein synthesis. (C) Quantitation of Aurora B protein levels in ARF^+/+ and ARF^−/− cells after cycloheximide treatment. Aurora B is substantially more stable in ARF^−/− than in ARF^+/+ cells. n = 3 independent experiments. Mean values for Aurora B protein levels are as follows (90% mean confidence intervals are shown in parentheses): ARF^+/+, 1 h, 0.7615 (0.5816, 0.9414); 2 h, 0.5503 (0.4635, 0.6371); 3 h, 0.6008 (0.4052, 0.7964); 6 h, 0.3308 (0.2618, 0.3997); 8 h, 0.4255 (0.3474, 0.5037); 12 h, 0.3417 (0.2518, 0.4315); ARF^−/−,1 h, 0.7777 (0.6665, 0.8889); 2 h, 0.9211 (0.8913, 0.951); 3 h, 1.061 (0.8448, 1.277); 6 h, 0.9289 (0.8282, 1.03); 8 h, 0.7647 (0.5965, 0.933); and 12 h, 0.5516 (0.2819, 0.8213).

FIGURE 8:

Overexpression of Aurora B phenocopies ARF loss. (A) Immunoblot showing expression of Aurora B-YFP in primary MEFs. Tubulin is shown as a loading control. (B, C) Aurora B-YFP localizes appropriately. Aurora B-YFP transiently transfected into wild-type MEFs localizes to (B) inner centromeres in prometaphase and (C) the midbody in telophase. Scale bars, 5 μm. (D) Reduced mitotic index in wild-type cells expressing Aurora B-YFP after 16 h of treatment with 100 ng/ml colcemid, showing that overexpression of Aurora B is sufficient to weaken mitotic checkpoint signaling. n > 250 cells from each of three independent experiments. (E–I) Wild-type MEFs transfected with Aurora B-YFP have elevated levels of (E) misaligned chromosomes in metaphase (n ≥ 50 cells with metaphase plates from three independent experiments), (F) lagging chromosomes in anaphase and telophase (n > 100 anaphases and telophases from three independent experiments), (G) binucleate interphase cells (n > 250 cells from each of three independent experiments), (H) supernumery (>2) centrosomes in interphase (n > 250 cells from each of three independent experiments), and (I) multipolar spindles in mitosis (n > 100 mitotic cells from each of three independent experiments), as compared with wild-type MEFs transfected with empty vector (EV). *p < 0.05.

FIGURE 9:

Partial knockdown of Aurora B to near wild-type levels rescues mitotic defects in ARF^−/− cells. (A) Immunoblot demonstrating partial knockdown of Aurora B in ARF^−/− primary MEFs to levels similar to those found in wild-type MEFs. Tubulin is shown as a loading control. (B) Partial knockdown of Aurora B rescues the mitotic checkpoint defect in ARF^−/− cells. n > 250 cells from each of three independent experiments. (C) Partial depletion of Aurora B by siRNA rescues the chromosome alignment defect in ARF^−/− cells. n > 50 cells with visible metaphase plates from each of three independent experiments. (D) Knockdown of Aurora B by siRNA to near-wild-type levels in ARF^−/− cells partially rescues the occurrence of lagging chromosomes. n > 100 anaphases and telophases from each of three independent experiments. *p < 0.05.