Cold fission: splitting the pombe cell at room temperature.

The mechanisms responsible for cytokinesis and its coordination with other events of the cell cycle are poorly understood. Genetic studies of cytokinesis in fission yeast are one useful approach to this problem. A number of conditional mutants of fission yeast that show defects in the formation of the septum of cytokinesis have been identified. Cloning of the genes affected in these mutants has begun to shed light upon the elements required to direct the construction of the division septum and also upon how the initiation of septum formation may be coordinated with mitosis.

The S. pombe cdc16 gene is required both for maintenance of p34cdc2 kinase activity and regulation of septum formation: a link between mitosis and cytokinesis?

In the fission yeast Schizosaccharomyces pombe, septum formation and cytokinesis are dependent upon the initiation, though not the completion of mitosis. A number of cell cycle mutants which show phenotypes consistent with a defect in the regulation of septum formation have been isolated. A mutation in the S. pombe cdc16 gene leads to the formation of multiple septa without cytokinesis, suggesting that the normal mechanisms that limit the cell to the formation of a single septum in each cycle do not operate. Mutations in the S. pombe early septation mutants cdc7, cdc11, cdc14 and cdc15 lead to the formation of elongated, multinucleate cells, as a result of S phase and mitosis continuing in the absence of cytokinesis. This suggests that in these cells, the normal mechanisms which initiate cytokinesis are defective and that they are unable to respond to this by preventing further nuclear cycles. Genetic analysis has implied that the products of some of these genes may interact with that of the cdc16 gene. To understand how the processes of septation and cytokinesis are regulated and coordinated with mitosis we are studying the early septation mutants and cdc16. In this paper, we present the cloning and analysis of the cdc16 gene. Deletion of the gene shows that it is essential for cell proliferation: spores lacking a functional cdc16 gene germinate, complete mitosis and form multiple septa without undergoing cell cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)

The dynamics of yeast telomeres and silencing proteins through the cell cycle.

Genes integrated near the telomeres of budding yeast have a variegated pattern of gene repression that is mediated by the silent information regulatory proteins Sir2p, Sir3p, and Sir4p. Immunolocalization and fluorescence in situ hybridization (FISH) reveal 6-10 perinuclear foci in which silencing proteins and subtelomeric sequences colocalize, suggesting that these are sites of Sir-mediated repression. Telomeres lacking subtelomeric repeat elements and the silent mating locus, HML, also localize to the periphery of the nucleus. Conditions that disrupt telomere proximal repression disrupt the focal staining pattern of Sir proteins, but not necessarily the localization of telomeric DNA. To monitor the telomere-associated pools of heterochromatin-binding proteins (Sir and Rap1 proteins) during mitotic cell division, we have performed immunofluorescence and telomeric FISH on populations of yeast cells synchronously traversing the cell cycle. We observe a partial release of Rap1p from telomeres in late G2/M, although telomeres appear to stay clustered during G2-phase and throughout mitosis. A partial release of Sir3p and Sir4p during mitosis also occurs. This is not observed upon HU arrest, although other types of DNA damage cause a dramatic relocalization of Sir and Rap1 proteins. The observed cell cycle dynamics were confirmed by direct epifluorescence of a GFP-Rap1p fusion. Using live GFP fluorescence we show that the diffuse mitotic distribution of GFP-Rap1p is restored to the interphase pattern of foci in early G1-phase.

Polar gradients of the DYRK-family kinase Pom1 couple cell length with the cell cycle.

Cells normally grow to a certain size before they enter mitosis and divide. Entry into mitosis depends on the activity of Cdk1, which is inhibited by the Wee1 kinase and activated by the Cdc25 phosphatase. However, how cells sense their size for mitotic commitment remains unknown. Here we show that an intracellular gradient of the dual-specificity tyrosine-phosphorylation regulated kinase (DYRK) Pom1, which emanates from the ends of rod-shaped Schizosaccharomyces pombe cells, serves to measure cell length and control mitotic entry. Pom1 provides positional information both for polarized growth and to inhibit cell division at cell ends. We discovered that Pom1 is also a dose-dependent G2-M inhibitor. Genetic analyses indicate that Pom1 negatively regulates Cdr1 and Cdr2, two previously described Wee1 inhibitors of the SAD kinase family. This inhibition may be direct, because in vivo and in vitro evidence suggest that Pom1 phosphorylates Cdr2. Whereas Cdr1 and Cdr2 localize to a medial cortical region, Pom1 forms concentration gradients from cell tips that overlap with Cdr1 and Cdr2 in short cells, but not in long cells. Disturbing these Pom1 gradients leads to Cdr2 phosphorylation and imposes a G2 delay. In short cells, Pom1 prevents precocious M-phase entry, suggesting that the higher medial Pom1 levels inhibit Cdr2 and promote a G2 delay. Thus, gradients of Pom1 from cell ends provide a measure of cell length to regulate M-phase entry.

The S. pombe cdc15 gene is a key element in the reorganization of F-actin at mitosis.

The S. pombe cdc15 gene is essential for cell division. cdc15ts mutants do not form a septum, but growth and nuclear division continue, leading to formation of multinucleate cells. The earliest step in septum formation and cytokinesis, rearrangement of actin to the center of the cell, is associated with appearance of hypophosphorylated cdc15p and formation of a cdc15p ring, which colocalizes with actin. Loss of cdc15p function impairs formation of the actin ring. The abundance of cdc15 mRNA varies through the cell division cycle, peaking in early mitosis before septation. Expression of cdc15 in G2-arrested cells induces actin rearrangement to the center of the cell. These data implicate cdc15p as a key element in mediating the cytoskeletal rearrangements required for cytokinesis.

An essential role for the Leishmania major metacaspase in cell cycle progression.

Metacaspases (MCAs) are distant orthologues of caspases and have been proposed to play a role in programmed cell death in yeast and plants, but little is known about their function in parasitic protozoa. The MCA gene of Leishmania major (LmjMCA) is expressed in actively replicating amastigotes and procyclic promastigotes, but at a lower level in metacyclic promastigotes. LmjMCA has a punctate distribution throughout the cell in interphase cells, but becomes concentrated in the kinetoplast (mitochondrial DNA) at the time of the organelle's segregation. LmjMCA also translocates to the nucleus during mitosis, where it associates with the mitotic spindle. Overexpression of LmjMCA in promastigotes leads to a severe growth retardation and changes in ploidy, due to defects in kinetoplast segregation and nuclear division and an impairment of cytokinesis. LmjMCA null mutants could not be generated and following genetic manipulation to express LmjMCA from an episome, the only mutants that were viable were those expressing LmjMCA at physiological levels. Together these data suggest that in L. major active LmjMCA is essential for the correct segregation of the nucleus and kinetoplast, functions that could be independent of programmed cell death, and that the amount of LmjMCA is crucial. The absence of MCAs from mammals makes the enzyme a potential drug target against protozoan parasites.

Inhibition of protein kinase B activity induces cell cycle arrest and apoptosis during early G₁ phase in CHO cells.

Inhibition of PKB (protein kinase B) activity using a highly selective PKB inhibitor resulted in inhibition of cell cycle progression only if cells were in early G1 phase at the time of addition of the inhibitor, as demonstrated by time-lapse cinematography. Addition of the inhibitor during mitosis up to 2 h after mitosis resulted in arrest of the cells in early G1 phase, as deduced from the expression of cyclins D and A and incorporation of thymidine. After 24 h of cell cycle arrest, cells expressed the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PKB activity in early G1 phase is required to prevent the induction of apoptosis. Using antibodies, it was demonstrated that active PKB translocates to the nucleus during early G1 phase, while an even distribution of PKB was observed through cytoplasm and nucleus during the end of G1 phase.

A 2D/3D image analysis system to track fluorescently labeled structures in rod-shaped cells: application to measure spindle pole asymmetry during mitosis.

BACKGROUND: The yeast Schizosaccharomyces pombe is frequently used as a model for studying the cell cycle. The cells are rod-shaped and divide by medial fission. The process of cell division, or cytokinesis, is controlled by a network of signaling proteins called the Septation Initiation Network (SIN); SIN proteins associate with the SPBs during nuclear division (mitosis). Some SIN proteins associate with both SPBs early in mitosis, and then display strongly asymmetric signal intensity at the SPBs in late mitosis, just before cytokinesis. This asymmetry is thought to be important for correct regulation of SIN signaling, and coordination of cytokinesis and mitosis. In order to study the dynamics of organelles or large protein complexes such as the spindle pole body (SPB), which have been labeled with a fluorescent protein tag in living cells, a number of the image analysis problems must be solved; the cell outline must be detected automatically, and the position and signal intensity associated with the structures of interest within the cell must be determined. RESULTS: We present a new 2D and 3D image analysis system that permits versatile and robust analysis of motile, fluorescently labeled structures in rod-shaped cells. We have designed an image analysis system that we have implemented as a user-friendly software package allowing the fast and robust image-analysis of large numbers of rod-shaped cells. We have developed new robust algorithms, which we combined with existing methodologies to facilitate fast and accurate analysis. Our software permits the detection and segmentation of rod-shaped cells in either static or dynamic (i.e. time lapse) multi-channel images. It enables tracking of two structures (for example SPBs) in two different image channels. For 2D or 3D static images, the locations of the structures are identified, and then intensity values are extracted together with several quantitative parameters, such as length, width, cell orientation, background fluorescence and the distance between the structures of interest. Furthermore, two kinds of kymographs of the tracked structures can be established, one representing the migration with respect to their relative position, the other representing their individual trajectories inside the cell. This software package, called "RodCellJ", allowed us to analyze a large number of S. pombe cells to understand the rules that govern SIN protein asymmetry. CONCLUSIONS: "RodCell" is freely available to the community as a package of several ImageJ plugins to simultaneously analyze the behavior of a large number of rod-shaped cells in an extensive manner. The integration of different image-processing techniques in a single package, as well as the development of novel algorithms does not only allow to speed up the analysis with respect to the usage of existing tools, but also accounts for higher accuracy. Its utility was demonstrated on both 2D and 3D static and dynamic images to study the septation initiation network of the yeast Schizosaccharomyces pombe. More generally, it can be used in any kind of biological context where fluorescent-protein labeled structures need to be analyzed in rod-shaped cells. AVAILABILITY: RodCellJ is freely available under http://bigwww.epfl.ch/algorithms.html, (after acceptance of the publication).

Responsiveness of astrocytes in serum-free aggregate cultures to epidermal growth factor: dependence on the cell cycle and the epidermal growth factor concentration.

Serum-free aggregating cell cultures of fetal rat telencephalon treated with low doses (0.5 nM) of epidermal growth factor (EGF) showed a small, transient increase in DNA synthesis but no significant changes in total DNA and protein content. By contrast, treatment with high doses (13 nM) of EGF caused a marked stimulation of DNA synthesis as well as a net increase in DNA and protein content. The expression of the astrocyte-specific enzyme, glutamine synthetase, was greatly enhanced both at low and at high EGF concentrations. These results suggest that at low concentration EGF stimulates exclusively the differentiation of astrocytes, whereas at high concentration, EGF has also a mitogenic effect. Nonproliferating astrocytes in cultures treated with 0.4 microM 1-beta-D-arabinofuranosyl-cytosine were refractory to EGF treatment, indicating that their responsiveness to EGF is cell cycle-dependent. Binding studies using a crude membrane fraction of 5-day cultures showed a homogeneous population of EGF binding sites (Kd approximately equal to 2.6 nM). Specific EGF binding sites were found also in non-proliferating (and nonresponsive) cultures, although they showed slightly reduced affinity and binding capacity. This finding suggests that the cell cycle-dependent control of astroglial responsiveness to EGF does not occur at the receptor level. However, it was found that the specific EGF binding sites disappear with progressive cellular differentiation.

Geometric control of the cell cycle.

How do cells sense their own size and shape? And how does this information regulate progression of the cell cycle? Our group, in parallel to that of Paul Nurse, have recently demonstrated that fission yeast cells use a novel geometry-sensing mechanism to couple cell length perception with entry into mitosis. These rod-shaped cells measure their own length by using a medially-placed sensor, Cdr2, that reads a protein gradient emanating from cell tips, Pom1, to control entry into mitosis. Budding yeast cells use a similar molecular sensor to delay entry into mitosis in response to defects in bud morphogenesis. Metazoan cells also modulate cell proliferation in response to their own shape by sensing tension. Here I discuss the recent results obtained for the fission yeast system and compare them to the strategies used by these other organisms to perceive their own morphology.

The activity of S.pombe DSC-1-like factor is cell cycle regulated and dependent on the activity of p34cdc2.

In the eukaryotic cell cycle, there are major control points in late G2 to determine the timing of the initiation of mitosis, and in late G1, regulating entry into S phase. In yeasts, this latter control is called start. Traverse of the start control and progression to S phase is accompanied by an increase in the expression of some of the genes whose products are required for DNA synthesis. In Saccharomyces cerevisiae, the coordinate expression of these genes in late G1 is dependent on a cis-acting sequence element called the MluI cell cycle box (MCB). A transcription factor called DSC-1 binds these elements and mediates cell cycle regulated transcription, though it is unclear whether this is by cell cycle-dependent changes in its activity. A DSC-1-like factor has also been identified in the fission yeast S.pombe. This is composed of at least the products of the cdc10 and sct1/res1 genes, and binds to the promoters of genes whose expression increases prior to S phase. We demonstrate that p85cdc10 is a nuclear protein and that the activity of the S.pombe DSC-1 factor varies through the cell cycle; it is high in cells that have passed start, decreases at the time of anaphase, remains low during the pre-start phase of G1 and increases at the time of the next S phase. We also show that the reactivation in late G1 is dependent on the G1 form of p34cdc2.

Asymmetric cancer cell division regulated by AKT.

Human tumors often contain slowly proliferating cancer cells that resist treatment, but we do not know precisely how these cells arise. We show that rapidly proliferating cancer cells can divide asymmetrically to produce slowly proliferating "G0-like" progeny that are enriched following chemotherapy in breast cancer patients. Asymmetric cancer cell division results from asymmetric suppression of AKT/PKB kinase signaling in one daughter cell during telophase of mitosis. Moreover, inhibition of AKT signaling with small-molecule drugs can induce asymmetric cancer cell division and the production of slow proliferators. Cancer cells therefore appear to continuously flux between symmetric and asymmetric division depending on the precise state of their AKT signaling network. This model may have significant implications for understanding how tumors grow, evade treatment, and recur.

The role of PLK-1 in asynchronous cell division of two-cell stage "C. elegans" embryos

Summary Acquisition of lineage-specific cell cycle duration is an important feature of metazoan development. In Caenorhabditis a/egans, differences in cell cycle duration are already apparent in two-cell stage embryos, when the larger anterior blastomere AB divides before the smaller posterior blastomere P1. This time difference is under the control of anterior-posterior (A-P) polarity cues set by the PAR proteins. The mechanism by which these cues regulate the cell cycle machinery differentially in AB and P1 are incompletely understood. Previous work established that retardation of P1 cell division is due in part to preferential activation of an ATL1/CHK-1 dependent checkpoint in P1 but how the remaining time difference is controlled was not known at the onset of my work. The principal line of work in this thesis established that differential timing relies also on a mechanism that promotes mitosis onset preferentially in AB. The polo-like kinase PLK-1, a positive regulator of mitotic entry, is distributed in an asymmetric manner in two-cell stage embryos, with more protein present in AB than in P1. We find that PLK-1 asymmetry is regulated by anterior-posterior (A-P) polarity cues through preferential protein retention in the embryo anterior. Importantly, mild inactivation of plk-1 by RNAi delays entry into mitosis in P1 but not in AB, in a manner that is independent of ATL-1/CHK-1. Together, these findings favor a model in which differential timing of mitotic entry in C. elegans embryos relies on two complementary mechanisms: ATL-1/CHK-1 dependent preferential retardation in P1 and PLK-1 dependent preferential promotion in AB, which together couple polarity cues and cell cycle progression during early development. Besides analyzing PLK-1 asymmetry and its role in differential timing of two-cells stage embryos, we also characterized t2190, a mutant that exhibits reduced differential timing between AB and P1. We found this mutant to be a new allele of par-1. Additionally, we analyzed the role of NMY-2 in regulating the asynchrony of two-cell stage embryos, which may be uncoupled from its role in A-P polarity establishment and carried out a preliminary analysis of the mechanism underlying CDC-25 asymmetry between AB and P,. Overall, our works bring new insights into the mechanism controlling cell cycle progression in early C. elegans embryos. As most of the players important in C. elegans are conserved in other organisms, analogous mechanisms may be utilized in polarized cells of other species. Résumé Au cours du développement, les processus de division cellulaire sont régulés dans l'espace et le temps afin d'aboutir à la formation d'un organisme fonctionnel. Chez les Métazoaires, l'un des mécanismes de contrôle s'effectue au niveau de la durée du cycle cellulaire, celle-ci étant specifiée selon la lignée cellulaire. L'embryon du nématode Caenorhabditis elegans apparaît comme un excellent modèle d'étude de la régulation temporelle du cycle cellulaire. En effet, suite à la première division du zygote, l'embryon est alors composé de deux cellules de taille et d'identité différentes, appelées blastomères AB et P1. Ces deux cellules vont ensuite se diviser de manière asynchrone, le grand blastomère antérieur AB se divisant plus rapidement que le petit blastomère postérieur P1. Cette asynchronie de division est sous le contrôle des protéines PAR qui sont impliquées dans l'établissement de l'axe antéro-postérieur de l'embryon. A ce jour, les mécanismes moléculaires gouvernant ce processus d'asynchronie ne sont que partiellement compris. Des études menées précédemment ont établit que le retard de division observé dans le petit blastomère postérieur P1 était dû, en partie, à l'activation préférentielle dans cette cellule de ATL-1/CHK-1, protéines contrôlant la réponse à des erreurs dans le processus de réplication de l'ADN. L'analyse des autres mécanismes responsables de la différence temporelle d'entrée en mitose des deux cellules a été entreprise au cours de cette thèse. Nous avons considéré la possibilité que l'asynchronie de division était du à l'entrée préférentielle en mitose du grand blastomère AB. Nous avons établi que la protéine kinase PLK-1 (polo-like kinase 1), impliquée dans la régulation positive de la mitose, était distribuée de manière asymétrique dans l'embryon deux cellules. PLK-1 est en effet enrichi dans le blastomère AB. Cette localisation asymétrique de PLK-1 est sous le contrôle des protéines PAR et semble établie via une rétention de PLK-1 dans la cellule AB. Par ailleurs, nous avons démontré que l'inactivation partielle de plk-7 par interférence à ARN (RNAi) conduit à un délai de l'entrée en mitose de la cellule P1 spécifiquement, indépendamment des protéines régulatrices ATL-1/CHK-1. En conclusion, nous proposons un modèle de régulation temporelle de l'entrée en mitose dans l'embryon deux cellules de C. elegans basé sur deux mécanismes complémentaires. Le premier implique l'activation préférentielle des protéines ATL-1/CHK-1, et conduit à un retard d'entrée en mitose spécifiquement dans la cellule P1. Le second est basé sur la localisation asymétrique de la protéine kinase PLK-1 dans la cellule AB et induit une entrée précoce en mitose de cette cellule. Par ailleurs, nous avons étudié un mutant appelé t2190 qui réduit la différence temporelle d'entrée en mitose entre les cellules AB et P1. Nous avons démontré que ce mutant correspondait à un nouvel allèle du Bene par-1. De plus, nous avons analysé le rôle de NMY-2, une protéine myosine qui agit comme moteur moléculaire sur les filaments d'active; dans la régulation de l'asynchronie de division des blastomères AB et P1, indépendamment de sa fonction dans l'établissement de l'axe antéro-postérieur. Par ailleurs, nous avons commencé l'étude du mécanisme moléculaire régulant la localisation asymétrique entre les cellules AB et P1 de la protéine phosphatase CDC25, qui est également un important régulateur de l'entrée en mitose. En conclusion, ce travail de thèse a permis une meilleure compréhension des mécanismes gouvernant la progression du cycle cellulaire dans l'embryon précoce de C. elegans. Etant donné que la plupart des protéines impliquées dans ces processus sont conservées chez d'autres organismes multicellulaires, il apparaît probable que les mécanismes moléculaires révélés dans cette étude soit aussi utilisés chez ceux-ci.

The DNA damage response to adeno-associated virus : centrosome overduplication and induction of cell death independently of the spindle checkpoint

Summary: Adeno-associated virus type 2 (AAV2) is a small virus containing single-stranded DNA of approximately 4.7kb in size. Both ends of the viral genome are flanked with inverted terminal repeat sequences (ITRs), which serve as primers for viral replication. Previous work in our laboratory has shown that AAV2 DNA with ultraviolet radiation-generated crosslinks (UV-AAV2) provokes a DNA damage response in the host cell by mimicking a stalled replication fork. Infection of cells with UV-AAV2 leads to a p53-and Chk1-mediated cell cycle arrest at the G2/M border of the cell cycle. However, tumour cells lacking the tumour suppressor protein p53 cannot sustain this arrest and enter a prolonged impaired mitosis, the outcome of which is cell death. The aim of my thesis was to investigate how UV-inactivated AAV2 kilts p53-deficient cancer cells. I found that the UV-AAV2-induced DNA damage signalling induces centriole overduplication in infected cells. The virus is able to uncouple the centriole duplication cycle from the cell cycle, leading to amplified centrosome numbers. Chk1 colocalises with centrosomes in the infected cells and the centrosome overduplication is dependent on the presence of Chk1, as well as on the activities of ATR and Cdk kinases and on the G2 arrest. The UV-AAV2-induced DNA damage signalling inhibits the degradation of cyclin B 1 and securin by the anaphase promoting complex, suggesting that the spindle checkpoint is activated in these mitotic cells. Interference with the spindle checkpoint components Mad2 and BubR1 revealed that the UV-AAV2-provoked mitotic catastrophe occurs independently of spindle checkpoint function, This work shows that, in the p53 deficient cells, UV-AAV2 triggers mitotic catastrophe associated with a dramatic Chk1-dependent overduplication of centrioles and the consequent formation of multiple spindle poles in mitosis. Résumé Le virus associé à l'adénovirus type 2 (AAV2) est un petit virus contenant un simple brin d'ADN d'environ 4.7kb. Des expériences antérieures dans notre laboratoire ont montré que les liens intramoléculaires sur l'ADN de AAV2 provoqués paz l'irradiation aux ultraviolets (UV) ressemblent à une fourche de réplication bloquée, ce qui provoque une réponse aux dommages à l'ADN dans la cellule hôte. L'infection des cellules avec UV-AAV2 résulte en un arrêt du cycle cellulaire à la transition G2/M entraîné par les protéines ATR et Chk1. Cependant, les cellules tumorales auxquelles il manque le suppresseur de tumeur p53 ne peuvent pas tenir cet arrêt et entrent dans une mitose anormale et prolongée qui se terminera par la mort cellulaire. Le but de ma thèse était d'étudier comment l'AAV2 inactivé par l'irradiation UV tue les cellules cancéreuses n'ayant pas p53. Je montre ici que le signal de dommages à l'ADN induit par UV-AAV2 génère une surduplication des centrioles dans les cellules infectées. Le virus est capable de dissocier le cycle de duplication du centriole du cycle cellulaire ce qui crée un nombre amplifié de centrosomes. Chk1 est co-localisé avec le centrosome dans les cellules infectées et la swduplication du centrosome est dépendante de la présence de Chk1, de l'activité des kinases ATR et Cdk et de l'arrêt en G2 de la cellule. Le signal d'ADN endommagé induit par UV-AAV2 réprime la dégradation des protéines cycline B1 et securine par le complexe promoteur de l'anaphase (APC), ce qui suggère que le point de contrôle du fuseau mitotique est activé dans ces cellules en mitose. L'étude d'interférence avec des éléments du point de contrôle du fuseau mitotique, Mad2 et BubR1, a révélé que la catastrophe mitotique provoquée paz UV-AAV2 survient indépendamment du point de contrôle du fuseau mitotique. Ce travail montre que dans les cellules déficientes en p53, UV-AAV2 induit une catastrophe mitotique associée à une surduplication des centrioles dépendant de Chk1 et ayant pour conséquence dramatique la formation de multiples fuseaux mitotiques dans la cellule en mitose.

The TRAF-interacting protein (TRAIP) is a novel E2F target with peak expression in mitosis.

The TRAF-interacting protein (TRAIP) is an E3 ubiquitin ligase required for cell proliferation. TRAIP mRNA is downregulated in human keratinocytes after inhibition of the PI3K/AKT/mTOR signaling. Since E2F transcription factors are downstream of PI3K/AKT/mTOR we investigated whether they regulate TRAIP expression. E2F1 expression significantly increased the TRAIP mRNA level in HeLa cells. Reporter assays with the 1400bp 5'-upstream promoter in HeLa cells and human keratinocytes showed that E2F1-, E2F2- and E2F4-induced upregulation of TRAIP expression is mediated by 168bp upstream of the translation start site. Mutating the E2F binding site within this fragment reduced the E2F1- and E2F2-dependent promoter activities and protein-DNA complex formation in gel shift assays. Abundance of TRAIP mRNA and protein was regulated by the cell cycle with a peak in G2/M. Expression of GFP and TRAIP-GFP demonstrated that TRAIP-GFP protein has a lower steady-state concentration than GFP despite similar mRNA levels. Cycloheximide inhibition experiments indicated that the TRAIP protein has a half-life of around four hours. Therefore, the combination of cell cycle-dependent transcription of the TRAIP gene by E2F and rapid protein degradation leads to cell cycle-dependent expression with a maximum in G2/M. These findings suggest that TRAIP has important functions in mitosis and tumorigenesis.