The cell cycle program of polypeptide labeling in Chlamydomonas reinhardtii

The cell cycle program of polypeptide labeling in syndhronous cultures of wild-type Chlamydomonas reinhardtii was analyzed by pulse-labeling cells with 35SO4 = or [3H]arginine at different cell cycle stages. Nearly 100 labeled membrane and soluble polypeptides were resolved and studied using one-dimensional sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis. The labeling experiments produced the following results. (a) Total 35SO4 = and [3H]arginine incorporation rates varied independently throughout the cell cycle. 35SO4 = incorporation was highest in the mid-light phase, while [3H]arginine incorporation peaked in the dark phase just before cell division. (b) The relative labeling rate for 20 of 100 polypeptides showed significant fluctuations (3-12 fold) during the cell cycle. The remaining polypeptides were labeled at a rate commensurate with total 35SO4 = or [3H]arginine incorporation. The polypeptides that showed significant fluctuations in relative labeling rates served as markers to identify cell cycle stages. (c) The effects of illumination conditions on the apparent cell cycle stage-specific labeling of polypeptides were tested. Shifting light-grown asynchronous cells to the dark had an immediate and pronounced effect on the pattern of polypeptide labeling, but shifting dark-phase syndhronous cells to the light had little effect. The apparent cell cycle variations in the labeling of ribulose 1,5-biphosphate (RUBP)-carboxylase were strongly influenced by illumination effects. (d) Pulse-chase experiments with light-grown asynchronous cells revealed little turnover or inter- conversion of labeled polypeptides within one cell generation, meaning that major polypeptides, whether labeled in a stage-specific manner or not, do not appear transiently in the cell cycle of actively dividing, light-grown cells. The cell cycle program of labeling was used to analyze effects of a temperature-sensitive cycle blocked (cb) mutant. A synchronous culture of ts10001 was shifted to restrictive temperature before its block point to prevent it from dividing. The mutant continued its cell cycle program of polypeptide labeling for over a cell generation, despite its inability to divide

The contribution of age structure to cell population responses to targeted therapeutics

Cells grown in culture act as a model system for analyzing the effects of anticancer compounds, which may affect cell behavior in a cell cycle position-dependent manner. Cell synchronization techniques have been generally employed to minimize the variation in cell cycle position. However, synchronization techniques are cumbersome and imprecise and the agents used to synchronize the cells potentially have other unknown effects on the cells. An alternative approach is to determine the age structure in the population and account for the cell cycle positional effects post hoc. Here we provide a formalism to use quantifiable age distributions from live cell microscopy experiments to parameterize an age-structured model of cell population response

Multiscale modelling of cancer progression and treatment control : the role of intracellular heterogeneities in chemotherapy treatment

Cancer is a complex, multiscale process involving interactions at intracellular, intercellular and tissue scales that are in turn susceptible to microenvironmental changes. Each individual cancer cell within a cancer cell mass is unique, with its own internal cellular pathways and biochemical interactions. These interactions contribute to the functional changes at the cellular and tissue scale, creating a heterogenous cancer cell population. Anticancer drugs are effective in controlling cancer growth by inflicting damage to various target molecules and thereby triggering multiple cellular and intracellular pathways, leading to cell death or cell-cycle arrest. One of the major impediments in the chemotherapy treatment of cancer is drug resistance driven by multiple mechanisms, including multi-drug and cell-cycle mediated resistance to chemotherapy drugs. In this article, we discuss two hybrid multiscale modelling approaches, incorporating multiple interactions involved in the sub-cellular, cellular and microenvironmental levels to study the effects of cell-cycle, phase-specific chemotherapy on the growth and progression of cancer cells.PostprintPeer reviewe

Epidermal growth factor-mediated T-cell factor/lymphoid enhancer factor transcriptional activity is essential but not sufficient for cell cycle progression in nontransformed mammary epithelial cells

Because beta-catenin target genes such as cyclin D1 are involved in cell cycle progression, we examined whether beta-catenin has a more pervasive role in normal cell proliferation, even upon stimulation by non-Wnt ligands. Here, we demonstrate that epidermal growth factor (EGF) stimulates T-cell factor/lymphoid enhancer factor (Tcf/Lef) transcriptional activity in nontransformed mammary epithelial cells (MCF-10A) and that its transcriptional activity is essential for EGF-mediated progression through G(1)/S phase. Thus, expression of dominant-negative Tcf4 blocks EGF-mediated Tcf/Lef transcriptional activity and bromodeoxyuridine uptake. In fact, the importance of EGF-mediated Tcf/Lef transcriptional activity for cell cycle progression may lie further upstream at the G(1)/S phase transition. We demonstrate that dominant-negative Tcf4 inhibits a reporter of cyclin D1 promoter activity in a dose-dependent manner. Importantly, dominant-negative Tcf4 suppresses EGF- mediated cell cycle activity specifically by thwarting EGF- mediated Tcf/Lef transcriptional activity, not by broader effects on EGF signaling. Thus, although expression of dominant-negative Tcf4 blocks EGF- mediated TOPFLASH activation, it has no effect on either EGF receptor or ERK phosphorylation, further underscoring the fact that Tcf/ Lef-mediated transcription is essential for cell cycle progression, even when other pro-mitogenic signals are at normal levels. Yet, despite its essential role, Tcf/Lef transcriptional activity alone is not sufficient for cell cycle progression. Serum also stimulates Tcf/ Lef transcriptional activation in MCF-10A cells but is unable to promote DNA synthesis. Taken together, our data support a model wherein EGF promotes Tcf/ Lef transcriptional activity, and this signal is essential but not sufficient for cell cycle activity

Acanthamoeba induces cell-cycle arrest in host cells

Acanthamoeba can cause fatal granulomatous amoebic encephalitis (GAE) and eye keratitis. However, the pathogenesis and pathophysiology of these emerging diseases remain unclear. In this study, the effects of Acanthamoeba on the host cell cycle using human brain microvascular endothelial cells (HBMEC) and human corneal epithelial cells (HCEC) were determined. Two isolates of Acanthamoeba belonging to the T1 genotype (GAE isolate) and T4 genotype (keratitis isolate) were used, which showed severe cytotoxicity on HBMEC and HCEC, respectively. No tissue specificity was observed in their ability to exhibit binding to the host cells. To determine the effects of Acanthamoeba on the host cell cycle, a cell-cycle-specific gene array was used. This screened for 96 genes specific for host cell-cycle regulation. It was observed that Acanthamoeba inhibited expression of genes encoding cyclins F and G1 and cyclin-dependent kinase 6, which are proteins important for cell-cycle progression. Moreover, upregulation was observed of the expression of genes such as GADD45A and p130 Rb, associated with cell-cycle arrest, indicating cell-cycle inhibition. Next, the effect of Acanthamoeba on retinoblastoma protein (pRb) phosphorylation was determined. pRb is a potent inhibitor of G1-to-S cell-cycle progression; however, its function is inhibited upon phosphorylation, allowing progression into S phase. Western blotting revealed that Acanthamoeba abolished pRb phosphorylation leading to cell-cycle arrest at the G1-to-S transition. Taken together, these studies demonstrated for the first time that Acanthamoeba inhibits the host cell cycle at the transcriptional level, as well as by modulating pRb phosphorylation using host cell-signalling mechanisms. A complete understanding of Acanthamoeba–host cell interactions may help in developing novel strategies to treat Acanthamoeba infections

Axitinib induces senescence-associated cell death and necrosis in glioma cell lines: The proteasome inhibitor, bortezomib, potentiates axitinib-induced cytotoxicity in a p21(Waf/Cip1) dependent manner.

Glioblastoma is associated with a poor overall survival despite new treatment advances. Antiangiogenic strategies targeting VEGF based on tyrosine kinase inhibitors (TKIs) are currently undergoing extensive research for the treatment of glioma. Herein we demonstrated that the TKI axitinib induces DNA damage response (DDR) characterized by γ-H2AX phosphorylation and Chk1 kinase activation leading to G2/M cell cycle arrest and mitotic catastrophe in U87, T98 and U251 glioma cell lines. Moreover, we found that p21(Waf1/Cip1) increased levels correlates with induction of ROS and senescence-associated cell death in U87 and T98 cell lines, which are reverted by N-acetyl cysteine pretreatment. Conversely, U251 cell line showed a resistant phenotype in response to axitinib treatment, as evidenced by cell cycle arrest but no sign of cell death. The combinatorial use of axitinib with other therapies, with the aim of inhibiting multiple signaling pathways involved in tumor growth, can increase the efficiency of this TKI. Thus, we addressed the combined effects of axitinib with no toxic doses of the proteasome inhibitor bortezomib on the growth of U87 and T98 axitinib- sensitive and axitinib-resistant U251 cell lines. Compared to single treatments, combined exposure was more effective in inhibiting cell viability of all glioma cell lines, although with different cell death modalities. The regulation of key DDR and cell cycle proteins, including Chk1, γ-H2AX and p21(Waf1/Cip1) was also studied in glioma cell lines. Collectively, these findings provide new perspectives for the use of axitinib in combination with Bortezomib to overcome the therapy resistance in gliomas

Parallel Load Balancing Strategies for Ensembles of Stochastic Biochemical Simulations

The evolution of biochemical systems where some chemical species are present with only a small number of molecules, is strongly influenced by discrete and stochastic effects that cannot be accurately captured by continuous and deterministic models. The budding yeast cell cycle provides an excellent example of the need to account for stochastic effects in biochemical reactions. To obtain statistics of the cell cycle progression, a stochastic simulation algorithm must be run thousands of times with different initial conditions and parameter values. In order to manage the computational expense involved, the large ensemble of runs needs to be executed in parallel. The CPU time for each individual task is unknown before execution, so a simple strategy of assigning an equal number of tasks per processor can lead to considerable work imbalances and loss of parallel efficiency. Moreover, deterministic analysis approaches are ill suited for assessing the effectiveness of load balancing algorithms in this context. Biological models often require stochastic simulation. Since generating an ensemble of simulation results is computationally intensive, it is important to make efficient use of computer resources. This paper presents a new probabilistic framework to analyze the performance of dynamic load balancing algorithms when applied to large ensembles of stochastic biochemical simulations. Two particular load balancing strategies (point-to-point and all-redistribution) are discussed in detail. Simulation results with a stochastic budding yeast cell cycle model confirm the theoretical analysis. While this work is motivated by cell cycle modeling, the proposed analysis framework is general and can be directly applied to any ensemble simulation of biological systems where many tasks are mapped onto each processor, and where the individual compute times vary considerably among tasks

Rescue of DNA damage after constricted migration reveals a mechano-regulated threshold for cell cycle.

Migration through 3D constrictions can cause nuclear rupture and mislocalization of nuclear proteins, but damage to DNA remains uncertain, as does any effect on cell cycle. Here, myosin II inhibition rescues rupture and partially rescues the DNA damage marker γH2AX, but an apparent block in cell cycle appears unaffected. Co-overexpression of multiple DNA repair factors or antioxidant inhibition of break formation also exert partial effects, independently of rupture. Combined treatments completely rescue cell cycle suppression by DNA damage, revealing a sigmoidal dependence of cell cycle on excess DNA damage. Migration through custom-etched pores yields the same damage threshold, with ∼4-µm pores causing intermediate levels of both damage and cell cycle suppression. High curvature imposed rapidly by pores or probes or else by small micronuclei consistently associates nuclear rupture with dilution of stiff lamin-B filaments, loss of repair factors, and entry from cytoplasm of chromatin-binding cGAS (cyclic GMP-AMP synthase). The cell cycle block caused by constricted migration is nonetheless reversible, with a potential for DNA misrepair and genome variation

Short-term inhibition of TERT induces telomere length-independent cell cycle arrest and apoptotic response in EBV-immortalized and transformed B cells

open7siBesides its canonical role in stabilizing telomeres, telomerase reverse transcriptase (TERT) may promote tumorigenesis through extra-telomeric functions. The possible therapeutic effects of BIBR1532 (BIBR), a powerful TERT inhibitor, have been evaluated in different cellular backgrounds, but no data are currently available regarding Epstein-Barr virus (EBV)-driven B-cell malignancies. Our aim was to characterize the biological effects of TERT inhibition by BIBR on EBV-immortalized lymphoblastoid cell lines (LCLs) and fully transformed Burkitt's lymphoma (BL) cell lines. We found that BIBR selectively inhibits telomerase activity in TERT-positive 4134/Late and 4134/TERT+ LCLs and EBV-negative BL41 and EBV-positive BL41/B95.8 BL cell lines. TERT inhibition led to decreased cell proliferation, accumulation of cells in the S-phase and ultimately to increased apoptosis, compared with mock-treated control cells. All these effects occurred within 72 h and were not observed in BIBR-treated TERT-negative 4134/TERT- and U2OS cells. The cell cycle arrest and apoptosis, consequent upon short-term TERT inhibition, were associated with and likely dependent on the activation of the DNA damage response (DDR), highlighted by the increased levels of γH2AX and activation of ATM and ATR pathways. Analyses of the mean and range of telomere lengths and telomere dysfunction-induced foci indicated that DDR after short-term TERT inhibition was not related to telomere dysfunction, thus suggesting that TERT, besides stabilizing telomere, may protect DNA via telomere-independent mechanisms. Notably, TERT-positive LCLs treated with BIBR in combination with fludarabine or cyclophosphamide showed a significant increase in the number of apoptotic cells with respect to those treated with chemotherapeutic agents alone. In conclusion, TERT inhibition impairs cell cycle progression and enhances the pro-apoptotic effects of chemotherapeutic agents in TERT-positive cells. These results support new therapeutic applications of TERT inhibitors in EBV-driven B-cell malignancies.openCeleghin, Andrea; Giunco, Silvia; Freguja, Riccardo; Zangrossi, Manuela; Nalio, Silvia; Dolcetti, Riccardo; De Rossi, AnitaCeleghin, Andrea; Giunco, Silvia; Freguja, Riccardo; Zangrossi, Manuela; Nalio, Silvia; Dolcetti, Riccardo; DE ROSSI, Anit