Drug-induced cell cycle modulation leading to cell-cycle arrest, nuclear mis-segregation, or endoreplication

<p>Abstract</p> <p>Background</p> <p>Cancer cell responses to chemotherapeutic agents vary, and this may reflect different defects in DNA repair, cell-cycle checkpoints, and apoptosis control. Cytometry analysis only quantifies dye-incorporation to examine DNA content and does not reflect the biological complexity of the cell cycle in drug discovery screens.</p> <p>Results</p> <p>Using population and time-lapse imaging analyses of cultured immortalized cells expressing a new version of the fluorescent cell-cycle indicator, Fucci (<it>F</it>luorescent <it>U</it>biquitination-based <it>C</it>ell <it>C</it>ycle <it>I</it>ndicator), we found great diversity in the cell-cycle alterations induced by two anticancer drugs. When treated with etoposide, an inhibitor of DNA topoisomerase II, HeLa and NMuMG cells halted at the G₂/M checkpoint. HeLa cells remained there, but NMuMG cells then overrode the checkpoint and underwent nuclear mis-segregation or avoided the checkpoint and entered the endoreplication cycle in a drug concentration dependent manner. In contrast, an inhibitor of Cdk4 led to G₁arrest or endoreplication in NMuMG cells depending upon the initial cell-cycle phase of drug exposure.</p> <p>Conclusions</p> <p>Drug-induced cell cycle modulation varied not only between different cell types or following treatment with different drugs, but also between cells treated with different concentrations of the same drug or following drug addition during different phases of the cell cycle. By combining cytometry analysis with the Fucci probe, we have developed a novel assay that fully integrates the complexity of cell cycle regulation into drug discovery screens. This assay system will represent a powerful drug-discovery tool for the development of the next generation of anti-cancer therapies.</p

Tracking of Normal and Malignant Progenitor Cell Cycle Transit in a Defined Niche.

While implicated in therapeutic resistance, malignant progenitor cell cycle kinetics have been difficult to quantify in real-time. We developed an efficient lentiviral bicistronic fluorescent, ubiquitination-based cell cycle indicator reporter (Fucci2BL) to image live single progenitors on a defined niche coupled with cell cycle gene expression analysis. We have identified key differences in cell cycle regulatory gene expression and transit times between normal and chronic myeloid leukemia progenitors that may inform cancer stem cell eradication strategies

Gravity sensing in plant and animal cells

Gravity determines shape of body tissue and affects the functions of life, both in plants and animals. The cellular response to gravity is an active process of mechanotransduction. Although plants and animals share some common mechanisms of gravity sensing in spite of their distant phylogenetic origin, each species has its own mechanism to sense and respond to gravity. In this review, we discuss current understanding regarding the mechanisms of cellular gravity sensing in plants and animals. Understanding gravisensing also contributes to life on Earth, e.g., understanding osteoporosis and muscle atrophy. Furthermore, in the current age of Mars exploration, understanding cellular responses to gravity will form the foundation of living in space

A Cell/Cilia Cycle Biosensor for Single-Cell Kinetics Reveals Persistence of Cilia after G1/S Transition Is a General Property in Cells and Mice

The cilia and cell cycles are inextricably linked. Centrioles in the basal body of cilia nucleate the ciliary axoneme and sequester pericentriolar matrix (PCM) at the centrosome to organize the mitotic spindle. Cilia themselves respond to growth signals, prompting cilia resorption and cell cycle re-entry. We describe a fluorescent cilia and cell cycle biosensor allowing live imaging of cell cycle progression and cilia assembly and disassembly kinetics in cells and inducible mice. We define assembly and disassembly in relation to cell cycle stage with single-cell resolution and explore the intercellular heterogeneity in cilia kinetics. In all cells and tissues analyzed, we observed cilia that persist through the G1/S transition and into S/G2/M-phase. We conclude that persistence of cilia after the G1/S transition is a general property. This resource will shed light at an individual cell level on the interplay between the cilia and cell cycles in development, regeneration, and disease. The cilia and cell cycles are fundamental processes coupled through shared machinery. Ford et al. develop and characterize a multicistronic biosensor that can simultaneously label the cell and cilia cycles in mice, enabling live imaging studies of their kinetics

Mechanism of Cancer Cell Death Induced by Depletion of an Essential Replication Regulator

Background: Depletion of replication factors often causes cell death in cancer cells. Depletion of Cdc7, a kinase essential for initiation of DNA replication, induces cancer cell death regardless of its p53 status, but the precise pathways of cell death induction have not been characterized. Methodology/Principal Findings: We have used the recently-developed cell cycle indicator, Fucci, to precisely characterize the cell death process induced by Cdc7 depletion. We have also generated and utilized similar fluorescent cell cycle indicators using fusion with other cell cycle regulators to analyze modes of cell death in live cells in both p53-positive and-negative backgrounds. We show that distinct cell-cycle responses are induced in p53-positive and-negative cells by Cdc7 depletion. p53-negative cells predominantly arrest temporally in G2-phase, accumulating CyclinB1 and other mitotic regulators. Prolonged arrest at G2-phase and abrupt entry into aberrant M-phase in the presence of accumulated CyclinB1 are followed by cell death at the post-mitotic state. Abrogation of cytoplasmic CyclinB1 accumulation partially decreases cell death. The ATR-MK2 pathway is responsible for sequestration of CyclinB1 with 14-3-3s protein. In contrast, p53-positive cancer cells do not accumulate CyclinB1, but appear to die mostly through entry into aberrant S-phase after Cdc7 depletion. The combination of Cdc7 inhibition with known anti-cancer agents significantly stimulates cell death effects in cancer cells in a genotype-dependent manner, providing a strategic basis for future combination therapies

Two coral fluorescent proteins of distinct colors for sharp visualization of cell-cycle progression

We cloned and characterized two new coral fluorescent proteins: h2-3 and 1-41. h2-3 formed an obligate dimeric complex and exhibited bright green fluorescence. On the other hand, 1-41 formed a highly multimeric complex and exhibited dim red fluorescence. We engineered 1-41 into AzaleaB5, a practically useful red-emitting fluorescent protein for cellular labeling applications. We fused h2-3 and AzaleaB5 to the ubiquitination domains of human Geminin and Cdt1, respectively, to generate a new color variant of Fucci (Fluorescent Ubiquitination-based Cell-Cycle Indicator): Fucci5. We found Fucci5 provided more reliable nuclear labeling for monitoring cell-cycle progression than the 1st and 2nd generations that used mAG/mKO2 and mVenus/mCherry, respectively. Key words: fluorescent protein, cell cycle, time-lapse imaging, flow cytometr

Knockout of all ErbB-family genes delineates their roles in proliferation, survival, and migration

The ErbB-family receptors play pivotal roles in the proliferation, migration, and survival of epithelial cells. Because our knowledge on the ErbB-family receptors was obtained largely by the exogenous application of their ligands, it remains unknown to which extent each of the ErbB contributes to these outputs. We here knocked out each ErbB gene, various combinations of ErbB genes, or all in Madin-Darby canine kidney cells to delineate the contribution of each gene. ERK activation waves during collective cell migration were mediated primarily by ErbB1 and secondarily by the ErbB2/ErbB3 heterodimer. Either ErbB1 or the ErbB2/ErbB3 complex was sufficient for the G1/S progression. The saturation cell density was markedly reduced in cells deficient in all ErbB-proteins, but not cells retaining only ErbB2, which cannot bind to ligands. Thus, the ligand-independent ErbB2 activity is sufficient for preventing apoptosis at high cell density. In short, systematic knockout of ErbB-family genes delineated the roles of each ErbB receptor

APC(CDH1) targets MgcRacGAP for destruction in the late M phase.

Male germ cell RacGTPase activating protein (MgcRacGAP) is an important regulator of the Rho family GTPases--RhoA, Rac1, and Cdc42--and is indispensable in cytokinesis and cell cycle progression. Inactivation of RhoA by phosphorylated MgcRacGAP is an essential step in cytokinesis. MgcRacGAP is also involved in G1-S transition and nuclear transport of signal transducer and activator of transcription 3/5 (STAT3/5). Expression of MgcRacGAP is strictly controlled in a cell cycle-dependent manner. However, the underlying mechanisms have not been elucidated.Using MgcRacGAP deletion mutants and the fusion proteins of full-length or partial fragments of MgcRacGAP to mVenus fluorescent protein, we demonstrated that MgcRacGAP is degraded by the ubiquitin-proteasome pathway in the late M to G1 phase via APC(CDH1). We also identified the critical region for destruction located in the C-terminus of MgcRacGAP, AA537-570, which is necessary and sufficient for CDH1-mediated MgcRacGAP destruction. In addition, we identified a PEST domain-like structure with charged residues in MgcRacGAP and implicate it in effective ubiquitination of MgcRacGAP.Our findings not only reveal a novel mechanism for controlling the expression level of MgcRacGAP but also identify a new target of APC(CDH1). Moreover our results identify a C-terminal region AA537-570 of MgcRacGAP as its degron