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Human inhibitor of apoptosis protein (IAP) survivin participates in regulation of chromosome segregation and mitotic exit ¹

MARKO J. KALLIO², MIKKO NIEMINEN^*, and JOHN E. ERIKSSON

University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA;
^* Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, FIN-20521, Turku, Finland; and
Department of Biology, University of Turku, FIN-20014 Turku, Finland

²Correspondence: University of Oklahoma Health Sciences Center, 975 N.E. 10th St., Biomedical Research Center, Rm. 266, Oklahoma City, OK 73104, USA. E-mail: Marko-Kallio{at}ouhsc.edu

SPECIFIC AIM

The human inhibitor of apoptosis protein (IAP) survivin and its lower eukaryote homologues have been implicated in apoptosis and regulation of cell cycle. In this study, we have investigated mitotic role(s) of survivin by using survivin antisense oligonucleotides (FsO^-) and function blocking anti-survivin antibodies (s^-Ab), followed by fluorescence microscopy and live cell cinematography; we postulate that the function of survivin in dividing cells is integrated with the spindle checkpoint mechanism composed of mitotic regulators that monitor integrity of the genome.

PRINCIPAL FINDINGS

1. Survivin antisense oligonucleotides induce abortive mitosis and polyploidy
To investigate the effects of survivin inhibition on mitotic progression, we introduced fluorescein isothiocyanate (FITC)-conjugated scrambled control (FcO^-) or survivin (FsO^-) oligonucleotides into the HeLa and PtK1 cells by either a microinjection method or liposome-mediated DNA transfer. In HeLa cells transfected with the FsO^-, the endogenous survivin protein was reduced significantly in a concentration-dependent manner by 30-to-80% (P<0.05; 200 nM FsO^-, P<0.01; 400 nM FsO^-) whereas the FcO^- had no effect. Moreover, the percentage of abnormally large and micronucleated (MN) cells was significantly increased (P<0.01) in the FsO^- transfected PtK1 cell populations (polyploid 18.1±3.3, MN 11.5±3.5) compared with controls (polyploid 3.1±0.8, MN 2.5±1.5). Induction of polyploidy was confirmed with HeLa cells that were microinjected with FcO^- or FsO^-.

In the chemically M phase-arrested PtK1 cells, the percentage of FITC-positive cells with MN was significantly increased at 0, 2, 6 (P<0.05), and 8 h (P<0.01) in the FsO^- transfected cells compared with controls, indicating that the FsO^--treated cells were able to bypass the M phase block. We also determined how many nocodazole-treated, M phase-arrested cells showed signs of abortive mitosis such as decondensation of chromatin and formation of cleavage furrow at the various times. FsO^- targeted cells aborted M phase more frequently than control cells especially at 6 (P<0.01) and 8 h (P<0.05), when 19.0 ± 2.3% and 12.3 ± 3.5% of the FITC-positive cells were in the process of exiting M phase, respectively.

2. Anti-survivin antibody (s^-Ab) induces premature onset of anaphase and escape from chemically induced M phase block
To investigate the effects of perturbed survivin function during different mitotic phases, we used a microinjection technique with s^-Ab (Fig. 1 A). For control injections, we used KCl-PO₄ microinjection buffer (cBuf). The cells injected with the s^-Ab between late prophase and early prometaphase showed specific cell division defects. The cells either separated their sister chromatids prematurely at late prometaphase or spent a significantly shorter period at the metaphase stage (Fig. 1B , C ). Nine of 22 injected PtK1 cells initiated anaphase at late prometaphase before all the chromosomes had moved to the spindle equator. The remainder of the s^-Ab-injected PtK1 cells (n=13) spend a significantly shorter time at the metaphase stage than the controls (Fig. 1C ; 4.6±1.1 min vs. 8.6±1.9 min, P<0.01). Furthermore, the cells injected with s^-Ab at late prophase or early prometaphase started anaphase in an average 26.8 ± 5.3 min (range 17–34 min) after the nuclear envelope breakdown (NEB), which is significantly different from the controls (Fig. 1C ; 32.9±6.9 min, P<0.05). All s^-Ab-injected cells, including those experiencing premature onset of anaphase, exit mitosis normally without defects in cytokinesis.

View larger version (86K): [in this window] [in a new window]   Figure 1. Effects of s-Ab microinjection on progression of cell division. A) In mitotic (M) PtK1 extracts, the s-Ab recognizes a major band at 16 kDa. Microscope images show the immunofluorescent localization of survivin at the mitotic spindle poles (arrowheads). B) Time-lapse sequence of a PtK1 cell injected with s-Ab at early prometaphase. The chromosomes move toward the spindle equator; 15 min after the injection, all but one (arrowhead) chromosome had aligned at the equatorial plate. When the sister chromatids of the aligned chromosomes separate 22 min after the injection, the unaligned chromosome is still mal-oriented and both of its sister chromatids move to the same spindle pole. C) Diagram showing the duration of different mitotic phases in cBuf and s-Ab-injected PtK1 cells. The s-Ab injection did not affect the prometaphase chromosome movements, as the mean duration of the prometaphase (NEB-Meta) was the same as in the cBuf-injected cells. However, the duration of metaphase stage (Meta-Ana) and the average time from NEB to onset of anaphase were significantly shorter in the s-Ab-injected cells than controls.

To determine whether blocking of survivin function during mitotic arrest would affect the length of the metaphase delay, we injected nocodazole- or taxol-treated PtK1 cells with s^-Ab. All of the s^-Ab-injected cells (n=10) escaped the M phase block within 3 h of the injection (149.7±37.8 min) whereas all the control cells (n=10) remained arrested in M phase for at least 8 h. Cells treated with spindle poisons and injected with s^-Ab decondensed their chromosomes and returned to interphase without completing cytokinesis, and consequently formed micronucleated giant cells.

3. The 3F3/2 phosphoepitope is lost prematurely from the mitotic kinetochores in s^-Ab-injected PtK1 cells
One marker for the spindle checkpoint is the 3F3/2 phosphoepitope that responds to physical tension mediated by the opposing spindle microtubules at the sister kinetochores of a chromosome. In a normal cell, 3F3/2 epitopes are phosphorylated during prometaphase and localize to the kinetochores of chromosomes and to the spindle poles, but as the chromosomes move to the spindle equator, the 3F3/2 epitope is dephosphorylated and lost from the kinetochores (but not from the spindle poles), which marks deactivation of the spindle checkpoint. In the s^-Ab-injected PtK1 cells, the 3F3/2 phosphoepitope is lost prematurely from the kinetochores of prometaphase chromosomes whereas the fluorescence of the spindle pole located 3F3/2 signals did not change over time (Fig. 2 A). The kinetics of the loss of fluorescent 3F3/2 signals from the kinetochores corresponds well with the timing of the precocious separation of the sister chromatids. Loss of all kinetochore-bound 3F3/2 phosphoepitope from the s^-Ab-injected cells takes on average 14.0 ± 3.0 min after NEB, which is significantly shorter than 24.0 ± 4.0 min of cBuf-injected cells (Fig. 2B ; P<0.01).

View larger version (61K): [in this window] [in a new window]   Figure 2. 3F3/2 phosphoepitope is lost precociously from the kinetochores of PtK1 cells after introduction of s-Ab. A) Fluorescent micrographs depicting two PtK1 cells injected with s-Ab at early prometaphase and then allowed to progress in their division for 3 and 8 min, respectively, before fixation and immunolabeling. The cell fixed 3 min after injection had a normal 3F3/2-staining pattern with bright 3F3/2 signals at the kinetochores of unaligned chromosomes (some depicted with arrowheads) and spindle poles (arrows). The cell fixed 8 min after the injection had lost most of its kinetochore-bound 3F3/2 epitope, although it was still at prometaphase stage with many unaligned chromosomes. Only two faint 3F3/2 positive kinetochores could be seen (arrowheads). The spindle poles had normal 3F3/2 signals (arrows). In both cells, the injected s-Ab accumulated at the spindle poles (arrows in the rabbit IgG panel). B) Percentage of 3F3/2 positive kinetochores in cell populations injected with s-Ab or cBuf. Groups of 5 to 10 PtK1 cells were injected with the antibody at late prophase/early prometaphase and allowed to progress in their division for the times indicated before fixation and immunolabeling with anti-3F3/2 ascites and Crest anti-centromere sera. The percentage of 3F3/2 spots colocalizing with Crest signals was determined for each injected cell. (ND, not detected).

CONCLUSIONS

Here we show that perturbation of the survivin function by antisense targeting at G2 phase of the cell cycle causes cell division defects, resulting in aneuploidy and polyploidy. To investigate the possible mechanisms of the observed mitotic defects in detail, we microinjected early mitotic PtK1 cells with s^-Ab and followed them through mitosis. To our surprise, the s^-Ab-injected cells exhibited premature separation of sister chromatids but had no apparent difficulties in exiting mitosis. To our knowledge this is the first data implicating survivin in the actual process of chromosome segregation. Moreover, in the s^-Ab-injected cells, the kinetochore-bound 3F3/2 phosphoepitope was lost prematurely at late prometaphase, which indicates that the spindle checkpoint was affected in these cells. Finally, introduction of sO^- or s^-Ab into M phase arrested cells induced abortive mitosis, yielding aneuploidy and polyploidy. Together, these results propose a link between the survivin function and control of metaphase-anaphase transition by the spindle checkpoint.

A possible explanation for the different mitotic effects induced by survivin targeting is provided by the recent discovery of three human and murine survivin isoforms. These survivin isoforms may play multiple roles in regulating apoptosis and cell division in mammals (Fig. 3 ). The present results raise questions about the possible mitotic target(s) and the mitotic signaling cascades the survivin isoforms may participate in (Fig. 3) . Survivin shares some characteristics with classical M phase regulatory molecules. It transiently localizes to the kinetochores, centrosomes, and microtubules during mitosis and is destroyed by ubiquitin-mediated proteolysis in a cell cycle-dependent manner. Moreover, the survivin-like C. elegans BIR-1 protein interacts with aurora-like kinase AIR-2, a member of aurora/Ipl1 kinase family that has been implicated in cell division and chromosome segregation. In addition, aurora2 kinase associates with Cdc20, a regulator of anaphase-promoting complex/cyclosome (APC/C) that counteracts effects of Mad2, an inhibitor of APC/C activity. Introduction of anti-Mad2 antibodies into mitotic cells causes premature activation of APC/C and precocious onset of anaphase, an event similar to what we have observed after s^-Ab injections. It is tempting to speculate that the cell division defects we detect after perturbation of survivin function could be caused by an impact on the aurora/Ipl1 kinases or on the activity of the APC/C. Finally, survivin may regulate 3F3/2 phoshoepitope phosphorylation, as the dephosphorylation and consequent loss of the 3F3/2 phosphoepitope from the metaphase kinetochores mark deactivation of the checkpoint and activation of the APC/C, an event that occurred prematurely in s^-Ab-injected cells. These results suggest that survivin is a mitotic regulator and possible component of the spindle checkpoint machinery. Elucidation of the mechanisms by which survivin modulates activity of the spindle checkpoint will be valuable in understanding how aneuploidy contributes to malignant cell growth and tumorigenesis.

View larger version (35K): [in this window] [in a new window]   Figure 3. Possible targets of survivin isoforms during mitosis. The differentially expressed survivin isoforms may participate in regulation of various cell division events, including separation of sister chromatids and cytokinesis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0280fje; to cite this article, use FASEB J. (October 29, 2001) 10.1096/fj.01-0280fje

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