Regulation of the Bub2/Bfa1 GAP complex by Cdc5 and cell cycle checkpoints.

During mitosis, a ras-related GTPase (Tem1) binds GTP and activates a signal transduction pathway to allow mitotic exit. During most of the cell cycle, Tem1 function is antagonized by a GTPase-activating protein complex, Bfa1/Bub2. How the Bfa1/Bub2 complex is regulated is not well understood. We find that Polo/Cdc5 kinase acts upstream of Bfa1/Bub2 in the mitotic exit network. Cdc5 phosphorylates Bfa1 and acts to antagonize Bfa1 function to promote mitotic exit. Bfa1 is regulated by multiple cell cycle checkpoints. The spindle assembly and spindle orientation checkpoints inhibit Bfa1 phosphorylation. DNA damage does not inhibit Bfa1 phosphorylation and instead causes a Rad53- and Dun1-dependent modification of Bfa1. Regulation of Bfa1 may therefore be a key step controlled by multiple checkpoint pathways to ensure a mitotic arrest.     

The p53 tumor suppressor participates in multiple cell cycle checkpoints

The process of cell division is highly ordered and regulated. Checkpoints exist to delay progression into the next cell cycle phase only when the previous step is fully completed. The ultimate goal is to guarantee that the two daughter cells inherit a complete and faithful copy of the genome. Checkpoints can become activated due to DNA damage, exogenous stress signals, defects during the replication of DNA, or failure of chromosomes to attach to the mitotic spindle. Abrogation of cell cycle checkpoints can result in death for a unicellular organism or uncontrolled proliferation and tumorigenesis in metazoans (Nyberg et al., 2002). The tumor suppressor p53 plays a critical role in each of these cell cycle checkpoints and is reviewed here.     

The Chlamydomonas cell cycle is regulated by a light/dark-responsive cell-cycle switch

Cultures of the unicellular green alga Chlamydomonas reinhardii can be synchronized by light/dark cycling not only under photoautotrophic but also under mixotrophic growth conditions. We observed that cultures synchronized in the presence of acetate continue to divide synchronously for one cell-cycle period when transferred to heterotrophic growth conditions. This finding enabled us to investigate the differential effects of light on cell growth and cell division. When cells were exposed to continuous light at the beginning of the growth period they entered the division phase earlier than dark-grown cells as a consequence of an increased growth rate. Illumination at the end of the growth period, however, caused a considerable delay in cell division and an extended growth period. The light-induced delay in cell division was also observed in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II. This finding demonstrates that cell division is directly influenced by a light/dard-responsive cell-cycle switch rather than by light/dark-dependent changes in energy metabolism. The importance of this light/dark control to the regulation of the Chlamydomonas cell cycle was investigated in comparison with other control mechanisms (size control, time control). We found that the light/dard-responsive cell-cycle switch regulates the transition from G1-to S-phase. This control mechanism is effective in cells which have attained the commitment to at least one round of DNA replication and division but have not attained the maximal cell mass which initiates cell division in the light.Abbreviations dCTP deoxycytidine 5-triphosphate - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Proteolysis of the Caulobacter McpA chemoreceptor is cell cycle regulated by a ClpX-dependent pathway

Proteolysis is involved in cell differentiation and the progression through the cell cycle in Caulobacter crescentus. We have constitutively expressed the transmembrane chemoreceptor McpA from a multicopy plasmid to demonstrate that McpA degradation is modulated during the cell cycle. The level of McpA protein starts to decrease only when the swarmer cells differentiate into stalked cells. The reduction in McpA protein levels is maintained until the stalked cells develop into predivisional cells, at which point the level returns to that observed in swarmer cells. The cell-cycle-regulated degradation of McpA does not require the last 12 C-terminal amino acids, but it does require three amino acids (AAL) located 15 residues away from the C terminus. The ClpXP protease is essential in C. crescentus for viability, and thus, we tested McpA degradation in xylose conditional mutants. The effect on McpA degradation occurred within two generations from the start of ClpX depletion. The conditional mutants' growth rate was only slightly affected, suggesting that ClpX is directly involved in McpA proteolysis.     

Transcription of multiple cell wall protein-encoding genes in Saccharomyces cerevisiae is differentially regulated during the cell cycle

The yeast cell wall consists of an internal skeletal layer and an outside protein layer. The synthesis of both β-1,3-glucan and chitin, which together form the cell wall skeleton, is cell cycle-regulated. We show here that the expression of five cell wall protein-encoding genes (CWP1, CWP2, SED1, TIP1 and TIR1) is also cell cycle-regulated. TIP1 is expressed in G1 phase, CWP1, CWP2 and TIR1 are expressed in S/G2 phase, and SED1 in M phase. The data suggest that these proteins fulfil distinct functions in the cell wall.     

Linking DNA damage to cell cycle checkpoints

Cell cycle checkpoints constitute a network of signal transduction mechanisms to monitor DNA damage and replication and thereby regulate progression through the cell cycle. A series of events is triggered in cells upon DNA damage. Here we describe a framework for the understanding of the functions of the core components involved in the cell cycle response to DNA damage and the relevance to the origin of cancer.     

Chk1: a double agent in cell cycle checkpoints

Two cell cycle surveillance systems--the DNA damage checkpoint and the spindle checkpoint--guard against genomic instability. The protein kinase Chk1 is a well-established signal transducer in the DNA damage checkpoint. In this issue of Developmental Cell, Zachos et al.(2007) present evidence to indicate that Chk1 also plays a critical role in the spindle checkpoint, suggesting an interplay between the DNA damage and spindle checkpoints.     

Understanding the limitations of radiation-induced cell cycle checkpoints

The DNA damage response pathways involve processes of double-strand break (DSB) repair and cell cycle checkpoint control to prevent or limit entry into S phase or mitosis in the presence of unrepaired damage. Checkpoints can function to permanently remove damaged cells from the actively proliferating population but can also halt the cell cycle temporarily to provide time for the repair of DSBs. Although efficient in their ability to limit genomic instability, checkpoints are not foolproof but carry inherent limitations. Recent work has demonstrated that the G1/S checkpoint is slowly activated and allows cells to enter S phase in the presence of unrepaired DSBs for about 4-6?h post irradiation. During this time, only a slowing but not abolition of S-phase entry is observed. The G2/M checkpoint, in contrast, is quickly activated but only responds to a level of 10-20 DSBs such that cells with a low number of DSBs do not initiate the checkpoint or terminate arrest before repair is complete. Here, we discuss the limitations of these checkpoints in the context of the current knowledge of the factors involved. We suggest that the time needed to fully activate G1/S arrest reflects the existence of a restriction point in G1-phase progression. This point has previously been defined as the point when mitogen starvation fails to prevent cells from entering S phase. However, cells that passed the restriction point can respond to DSBs, albeit with reduced efficiency.     

DNA damage-dependent cell cycle checkpoints and genomic stability

In response to genotoxic stress, which can be caused by environmental or endogenous genotoxic insults such as ionizing or ultraviolet radiation, various chemicals and reactive cellular metabolites, cell cycle checkpoints which slow down or arrest cell cycle progression can be activated, allowing the cell to repair or prevent the transmission of damaged or incompletely replicated chromosomes. Checkpoint machineries can also initiate pathways leading to apoptosis and the removal of a damaged cell from a tissue. The balance between cell cycle arrest and damage repair on one hand and the initiation of cell death, on the other hand, could determine if cellular or DNA damage is compatible with cell survival or requires cell elimination by apoptosis. Defects in these processes may lead to hypersensitivity to cellular stress, and susceptibility to DNA damage, genomic defects, and resistance to apoptosis, which characterize cancer cells. In this article, we have noted recent studies of DNA damage-dependent cell cycle checkpoints, which may be significant in preventing genomic instability.     

Activation-induced T-cell death is cell cycle dependent and regulated by cyclin B.

Developing thymocytes and some T-cell hybridomas undergo activation-dependent programmed cell death. Although recent studies have identified some critical regulators in programmed cell death, the role of cell cycle regulation in activation-induced cell death in T cells has not been addressed. We demonstrate that synchronized T-cell hybridomas, irrespective of the point in the cell cycle at which they are activated, stop cycling shortly after they reach G2/M. These cells exhibit the diagnostic characteristics of apoptotic cell death. Although p34cdc2 levels are not perturbed after activation of synchronously cycling T cells, cyclin B- and p34cdc2-associated histone H1 kinase activity is persistently elevated. This activation-dependent induction of H1 kinase activity in T cells is associated with a decrease in the phosphotyrosine content of p34cdc2. We also demonstrate that transient inappropriate coexpression of cyclin B with p34cdc2 induces DNA fragmentation in a heterologous cell type. Finally, in T cells, cyclin B-specific antisense oligonucleotides suppress activation-induced cell death but not cell death induced by exposure to dexamethasone. We therefore conclude that a persistent elevation of the level of cyclin B kinase is required for activation-induced programmed T-cell death.     

CD18 is required for intestinal T cell responses at multiple immune checkpoints

The intestinal immune response to oral Ags involves a complex multistep process. The requirements for optimal intestinal T cell responses in this process are unclear. LFA-1 plays a critical role in peripheral T cell trafficking and activation, however, its role in intestinal immune responses has not been precisely defined. To dissect the role of LFA-1 in intestinal immune responses, we used a system that allows for segregation of T cell migration and activation through the adoptive transfer of LFA-1-deficient (CD18(-/-)) CD4(+) T cells from DO11.10 TCR transgenic mice into wild-type BALB/c mice. We find that wild-type mice adoptively transferred with CD18(-/-) DO11.10 CD4(+) T cells demonstrate decreases in the numbers of Ag-specific T cells in the intestinal lamina propria after oral Ag administration. We also find that in addition to its role in trafficking to intestinal secondary lymphoid organs, LFA-1 is required for optimal CD4(+) T cell proliferation in vivo upon oral Ag immunization. Furthermore, CD18(-/-) DO11.10 CD4(+) T cells primed in the intestinal secondary lymphoid organs demonstrate defects in up-regulation of the intestinal-specific trafficking molecules, alpha(4)beta(7) and CCR9. Interestingly, the defect in trafficking of CD18(-/-) DO11.10 CD4(+) T cells to the intestinal lamina propria persists even under conditions of equivalent activation and intestinal-tropic differentiation, implicating a role for CD18 in the trafficking of activated T cells into intestinal tissues independent of the earlier defects in the intestinal immune response. This argues for a complex role for CD18 in the early priming checkpoints and ultimately in the trafficking of T cells to the intestinal tissues during an intestinal immune response.     

Dose dependent effects on cell cycle checkpoints and DNA repair by bendamustine

Bendamustine (BDM) is an active chemotherapeutic agent approved in the U. S. for treating chronic lymphocytic leukemia and non-Hodgkin lymphoma. Its chemical structure suggests it may have alkylator and anti-metabolite activities; however the precise mechanism of action is not well understood. Here we report the concentration-dependent effects of BDM on cell cycle, DNA damage, checkpoint response and cell death in HeLa cells. Low concentrations of BDM transiently arrested cells in G2, while a 4-fold higher concentration arrested cells in S phase. DNA damage at 50, but not 200 μM, was efficiently repaired after 48 h treatment, suggesting a difference in DNA repair efficiency at the two concentrations. Indeed, perturbing base-excision repair sensitized cells to lower concentrations of BDM. Timelapse studies of the checkpoint response to BDM showed that inhibiting Chk1 caused both the S- and G2-arrested cells to prematurely enter mitosis. However, whereas the cells arrested in G2 (low dose BDM) entered mitosis, segregated their chromosomes and divided normally, the S-phase arrested cells (high dose BDM) exhibited a highly aberrant mitosis, whereby EM images showed highly fragmented chromosomes. The vast majority of these cells died without ever exiting mitosis. Inhibiting the Chk1-dependent DNA damage checkpoint accelerated the time of killing by BDM. Our studies suggest that BDM may affect different biological processes depending on drug concentration. Sensitizing cells to killing by BDM can be achieved by inhibiting base-excision repair or disrupting the DNA damage checkpoint pathway.     

Viral manipulation of DNA repair and cell cycle checkpoints

Recognition and repair of DNA damage is critical for maintaining genomic integrity and suppressing tumorigenesis. In eukaryotic cells, the sensing and repair of DNA damage are coordinated with cell cycle progression and checkpoints, in order to prevent the propagation of damaged DNA. The carefully maintained cellular response to DNA damage is challenged by viruses, which produce a large amount of exogenous DNA during infection. Viruses also express proteins that perturb cellular DNA repair and cell cycle pathways, promoting tumorigenesis in their quest for cellular domination. This review presents an overview of strategies employed by viruses to manipulate DNA damage responses and cell cycle checkpoints as they commandeer the cell to maximize their own viral replication. Studies of viruses have identified key cellular regulators and revealed insights into molecular mechanisms governing DNA repair, cell cycle checkpoints, and transformation.