Tamara A. Potapova

The Reversibility of Mitotic Exit in Vertebrate Cells

A guiding hypothesis for cell-cycle regulation asserts that regulated proteolysis constrains the directionality of certain cell-cycle transitions. Here we test this hypothesis for mitotic exit, which is regulated by degradation of the cyclin-dependent kinase 1 (Cdk1) activator, cyclin B. Application of chemical Cdk1 inhibitors to cells in mitosis induces cytokinesis and other normal aspects of mitotic exit, including cyclin B degradation. However, chromatid segregation fails, resulting in entrapment of chromatin in the midbody. If cyclin B degradation is blocked with a proteasome inhibitor or by expression of non-degradable cyclin B, Cdk inhibitors will nonetheless induce mitotic exit and cytokinesis. However, if after mitotic exit, the Cdk1 inhibitor is washed free from cells in which cyclin B degradation is blocked, the cells can revert back to M phase. This reversal is characterized by chromosome recondensation, nuclear envelope breakdown, assembly of microtubules into a mitotic spindle, and in most cases, dissolution of the midbody, reopening of the cleavage furrow, and realignment of chromosomes at the metaphase plate. These findings demonstrate that proteasome-dependent degradation of cyclin B provides directionality for the M phase to G1 transition.

Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited

Activation of Cdk1 is rapid and switch-like due to positive feedback mechanisms. When Cdk1 is fully on, cells are capable of M-to-G1 transition. Inhibition of positive feedback prevents rapid Cdk1 activation and induces a mitotic “collapse” phenotype characterized by the dephosphorylation of mitotic substrates without cyclin B proteolysis.

Fine Tuning the Cell Cycle: Activation of the Cdk1 Inhibitory Phosphorylation Pathway during Mitotic Exit

Inactivation of cyclin-dependent kinase (Cdk) 1 promotes exit from mitosis and establishes G1. Proteolysis of cyclin B is the major known mechanism that turns off Cdk1 during mitotic exit. Here, we show that mitotic exit also activates pathways that catalyze inhibitory phosphorylation of Cdk1, a mechanism previously known to repress Cdk1 only during S and G2 phases of the cell cycle. We present evidence that down-regulation of Cdk1 activates Wee1 and Myt1 kinases and inhibits Cdc25 phosphatase during the M to G1 transition. If cyclin B/Cdk1 complex is present in G1, the inhibitory sites on Cdk1 become phosphorylated. Exit from mitosis induced by chemical Cdk inhibition can be reversed if cyclin B is preserved. However, this reversibility decreases with time after mitotic exit despite the continued presence of the cyclin. We show that this G1 block is due to phosphorylation of Cdk1 on inhibitory residues T14 and Y15. Chemical inhibition of Wee1 and Myt1 or expression of Cdk1 phosphorylation site mutants allows reversal to M phase even from late G1. This late Cdk1 reactivation often results in caspase-dependent cell death. Thus, in G1, the Cdk inhibitory phosphorylation pathway is functional and can lock Cdk1 in the inactive state.

Aneuploidy and chromosomal instability: a vicious cycle driving cellular evolution and cancer genome chaos

Aneuploidy and chromosomal instability frequently co-exist, and aneuploidy is recognized as a direct outcome of chromosomal instability. However, chromosomal instability is widely viewed as a consequence of mutations in genes involved in DNA replication, chromosome segregation, and cell cycle checkpoints. Telomere attrition and presence of extra centrosomes have also been recognized as causative for errors in genomic transmission. Here, we examine recent studies suggesting that aneuploidy itself can be responsible for the procreation of chromosomal instability. Evidence from both yeast and mammalian experimental models suggests that changes in chromosome copy number can cause changes in dosage of the products of many genes located on aneuploid chromosomes. These effects on gene expression can alter the balanced stoichiometry of various protein complexes, causing perturbations of their functions. Therefore, phenotypic consequences of aneuploidy will include chromosomal instability if the balanced stoichiometry of protein machineries responsible for accurate chromosome segregation is affected enough to perturb the function. The degree of chromosomal instability will depend on specific karyotypic changes, which may be due to dosage imbalances of specific genes or lack of scaling between chromosome segregation load and the capacity of the mitotic system. We propose that the relationship between aneuploidy and chromosomal instability can be envisioned as a “vicious cycle,” where aneuploidy potentiates chromosomal instability leading to further karyotype diversity in the affected population.

Expression of HPV16 E5 produces enlarged nuclei and polyploidy through endoreplication

Anogenital cancers and head and neck cancers are causally associated with infection by high-risk human papillomavirus (HPV). The mechanism by which high-risk HPVs contribute to oncogenesis is poorly understood. HPV16 encodes three genes (HPV16 E5, E6, and E7) that can transform cells when expressed independently. HPV16 E6 and E7 have well-described roles causing genomic instability and unregulated cell cycle progression. The role of HPV16 E5 in cell transformation remains to be elucidated. Expression of HPV16 E5 results in enlarged, polyploid nuclei that are dependent on the level and duration of HPV16 E5 expression. Live cell imaging data indicate that these changes do not arise from cell-cell fusion or failed cytokinesis. The increase in nuclear size is a continual process that requires DNA synthesis. We conclude that HPV16 E5 produces polyploid cells by endoreplication. These findings provide insight into how HPV16 E5 can contribute to cell transformation.

The Consequences of Chromosome Segregation Errors in Mitosis and Meiosis

Mistakes during cell division frequently generate changes in chromosome content, producing aneuploid or polyploid progeny cells. Polyploid cells may then undergo abnormal division to generate aneuploid cells. Chromosome segregation errors may also involve fragments of whole chromosomes. A major consequence of segregation defects is change in the relative dosage of products from genes located on the missegregated chromosomes. Abnormal expression of transcriptional regulators can also impact genes on the properly segregated chromosomes. The consequences of these perturbations in gene expression depend on the specific chromosomes affected and on the interplay of the aneuploid phenotype with the environment. Most often, these novel chromosome distributions are detrimental to the health and survival of the organism. However, in a changed environment, alterations in gene copy number may generate a more highly adapted phenotype. Chromosome segregation errors also have important implications in human health. They may promote drug resistance in pathogenic microorganisms. In cancer cells, they are a source for genetic and phenotypic variability that may select for populations with increased malignance and resistance to therapy. Lastly, chromosome segregation errors during gamete formation in meiosis are a primary cause of human birth defects and infertility. This review describes the consequences of mitotic and meiotic errors focusing on novel concepts and human health.

Transcriptome analysis of tetraploid cells identifies cyclin D2 as a facilitator of adaptation to genome doubling in the presence of p53

Gene expression analysis indicates that p53-mediated suppression of proliferation of polyploid cells can be averted by increased levels of oncogenes such as cyclin D2. Tetraploid cells can adapt and continue to proliferate despite having increased genome content and a wild-type p53 signaling cascade.

Robust mitotic entry is ensured by a latching switch.

Summary Cell cycle events are driven by Cyclin dependent kinases (CDKs) and by their counter-acting phosphatases. Activation of the Cdk1:Cyclin B complex during mitotic entry is controlled by the Wee1/Myt1 inhibitory kinases and by Cdc25 activatory phosphatase, which are themselves regulated by Cdk1:Cyclin B within two positive circuits. Impairing these two feedbacks with chemical inhibitors induces a transient entry into M phase referred to as mitotic collapse. The pathology of mitotic collapse reveals that the positive circuits play a significant role in maintaining the M phase state. To better understand the function of these feedback loops during G2/M transition, we propose a simple model for mitotic entry in mammalian cells including spatial control over Greatwall kinase phosphorylation. After parameter calibration, the model is able to recapture the complex and non-intuitive molecular dynamics reported by Potapova et al. (Potapova et al., 2011). Moreover, it predicts the temporal patterns of other mitotic regulators which have not yet been experimentally tested and suggests a general design principle of cell cycle control: latching switches buffer the cellular stresses which accompany cell cycle processes to ensure that the transitions are smooth and robust.

Karyotyping human and mouse cells using probes from single-sorted chromosomes and open source software.

Multispectral karyotyping analyzes all chromosomes in a single cell by labeling them with chromosome-specific probes conjugated to unique combinations of fluorophores. Currently available multispectral karyotyping systems require the purchase of specialized equipment and reagents. However, conventional laser scanning confocal microscopes that are capable of separating multiple overlapping emission spectra through spectral imaging and linear unmixing can be utilized for classifying chromosomes painted with multicolor probes. Here, we generated multicolor chromosome paints from single-sorted human and mouse chromosomes and developed the Karyotype Identification via Spectral Separation (KISS) analysis package, a set of freely available open source ImageJ tools for spectral unmixing and karyotyping. Chromosome spreads painted with our multispectral probe sets can be imaged on widely available spectral laser scanning confocal microscopes and analyzed using our ImageJ tools. Together, our probes and software enable academic labs with access to a laser-scanning spectral microscope to perform multicolor karyotyping in a cost-effective manner.

Abstract 3868: Long-term inhibition of cells at metaphase with intact mitotic spindles results in unscheduled chromatid separation and catastrophic arrest in mitosis

In the presence of unaligned chromosomes or other spindle abnormalities, the mitotic spindle checkpoint blocks both chromatid separation at anaphase and exit from mitosis. The spindle checkpoint is sustained by signaling from kinetochores that are not properly attached to microtubules of the mitotic spindle. Normally, when chromosomes align at the spindle equator at metaphase, microtubule-kinetochore interactions act to extinguish checkpoint signaling. The cessation of the checkpoint signal activates the Anaphase-Promoting Complex/Cyclosome (APC/C), an E3 ubiquitin ligase. The APC/C ubiquitylates the mitotic effectors, securin and cyclin B, which are then degraded by the proteasome. Securin and cyclin B degradation initiates chromatid separation at anaphase and triggers mitotic exit. We have found that long-term arrest of Hela cells with their chromosomes aligned at the metaphase plate results in chromatid separation without mitotic exit. Recently, we and others have characterized C13orf3/Ska3, a newly discovered component of the Ska (Spindle and kinetochore-associated) protein complex. We found that in mitosis Ska3 protein accumulates at kinetochores reaching maximal levels at metaphase. Upon anaphase onset, kinetochore-associated Ska3 levels decline and are eventually lost at telophase. We demonstrated that cells depleted of Ska3 by RNAi achieve metaphase alignment but fail to silence the spindle checkpoint. During this mitotic arrest with chromosomes aligned at the metaphase plate, kinetochores retain robust microtubule attachments. Kinetochores within these Ska3-depleted cells accumulate abnormally high levels of the mitotic spindle checkpoint protein, Bub1, presumably accounting for the inability to silence the checkpoint. Under extended metaphase arrest, the chromatids of individual chromosomes eventually separate, but cells remain arrested in mitosis. To test if other means of inducing metaphase arrest would also lead to chromatid separation, we treated cells in mitosis with the proteasome inhibitor, MG132. Again, after several hours at metaphase, chromatids separated. We speculate that for cells arrested at metaphase, continual pulling forces of the kinetochores on the mitotic spindle eventually overcomes the cohesin complex that holds chromatids together. This “cohesin slippage” results in abnormal chromatid separation for cells still in mid-mitosis. This response reflects a novel terminal phenotype for cells arrested in mitosis with an intact mitotic spindle. (Supported by the NIGMS and the McCasland Foundation.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3868.