Mitotic Bypass Via An Occult Cell Cycle Phase Following DNA Topoisomerase II Inhibition In p53 Functional Human Tumor Cells

Smith, Paul J.; Márquez, Nuria Ponce; Wiltshire, Marie; Chappell, Sally Claire; Njoh, Kerenza; Campbell, Lee Ann; Khan, Imtiaz; Silvestre, Óscar F.; Errington, Rachel J.

doi:10.4161/cc.6.16.4585

Cited by 17 publications

(11 citation statements)

References 32 publications

(62 reference statements)

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“…Two hundred microliters of mouse immunoglobulin G in 0.5% BSA/PBS were added to the remaining coverslips at a matched concentration. After being left overnight at 4°C, coverslips were washed twice with PBS, and FITC was then added at a 1:75 or 1:100 dilution in 0.5% BSA/PBS, followed by incubation at 37°C, in air with 5% CO2, for 1 h. After coverslips were washed twice in PBS, coverslips were mounted on slides with Vectashield (Vector Laboratories) before imaging by laser scanning microscopy (42).…”

Section: Methodsmentioning

confidence: 99%

Impact of overexpression of metallothionein-1 on cell cycle progression and zinc toxicity

Smith

Wiltshire²,

Furon³

et al. 2008

American Journal of Physiology-Cell Physiology

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Metallothioneins (MTs) have an important role in zinc homeostasis and may counteract the impact of oversupply. Both intracellular zinc and MT expression have been implicated in proliferation control and resistance to cellular stress, although the interdependency is unclear. The study addresses the consequences of a steady-state overexpression of MT-1 for intracellular zinc levels, cell cycle progression, and protection from zinc toxicity using a panel of cell lines with differential expression of MT-1. The panel comprised parental Chinese hamster ovary-K1 cells with low endogenous expression of MT and transfectants with enhanced expression of mouse MT-1 on an autonomously replicating expression vector with a noninducible promoter. Cell cycle progression, determined by flow cytometry and time-lapse microscopy, revealed that enhanced cytoplasmic expression of MT-1 does not impact on normal cell cycle operation, suggesting that basal levels of MT-1 expression are not limiting for background levels of oxidative stress. MT-1 overexpression correlated with a steady-state increase in cytoplasmic free Zn(2+), assessed using the fluorescent zinc-sensor Zinquin, particularly at high levels of overexpression, further suggesting that zinc availability is normally not limiting for cell cycle progression. Enhanced MT-1 expression, over a 10-fold range, had a clear impact on resistance to Cd(2+) and Zn(2+) toxicity. In the case of Zn(2+), the degree of protection afforded was less, indicating that MT-1 has a limited range and saturable capacity for effecting resistance. The results have implications for the use of cellular stress responses to exogenously supplied zinc and zinc-based systemic therapies.

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Section: Methodsmentioning

confidence: 99%

Impact of overexpression of metallothionein-1 on cell cycle progression and zinc toxicity

Smith

Wiltshire²,

Furon³

et al. 2008

American Journal of Physiology-Cell Physiology

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“…To obtain fluorescence read-out of DNA content an anthraquinone derivative, DRAQ5™ (20 µM Biostatus Ltd., UK) was used [14]. This binds to DNA providing a fluorescence intensity that can be related to DNA content and thus it reports on cell cycle progression through the S phase to G 2 /M (>4N) or, in the presence of external perturbing agents, progression through polyploid states as the mitotic stage is by-passed [20] (see Figure 1(a)). To obtain a model system in which we can test the simulated cell population approach we have used a cell division by-pass agent: ICRF-193 [bis(2,6-dioxopiperazine)], a kind gift from Dr A.M. Creighton (ICRF, London, UK).…”

Section: Methodsmentioning

confidence: 99%

Flow-Based Cytometric Analysis of Cell Cycle via Simulated Cell Populations

et al. 2010

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We present a new approach to the handling and interrogating of large flow cytometry data where cell status and function can be described, at the population level, by global descriptors such as distribution mean or co-efficient of variation experimental data. Here we link the “real” data to initialise a computer simulation of the cell cycle that mimics the evolution of individual cells within a larger population and simulates the associated changes in fluorescence intensity of functional reporters. The model is based on stochastic formulations of cell cycle progression and cell division and uses evolutionary algorithms, allied to further experimental data sets, to optimise the system variables. At the population level, the in-silico cells provide the same statistical distributions of fluorescence as their real counterparts; in addition the model maintains information at the single cell level. The cell model is demonstrated in the analysis of cell cycle perturbation in human osteosarcoma tumour cells, using the topoisomerase II inhibitor, ICRF-193. The simulation gives a continuous temporal description of the pharmacodynamics between discrete experimental analysis points with a 24 hour interval; providing quantitative assessment of inter-mitotic time variation, drug interaction time constants and sub-population fractions within normal and polyploid cell cycles. Repeated simulations indicate a model accuracy of ±5%. The development of a simulated cell model, initialized and calibrated by reference to experimental data, provides an analysis tool in which biological knowledge can be obtained directly via interrogation of the in-silico cell population. It is envisaged that this approach to the study of cell biology by simulating a virtual cell population pertinent to the data available can be applied to “generic” cell-based outputs including experimental data from imaging platforms.

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“…NBCs can divide an unlimited number of times (theoretically) but rarely differentiate in the presence of an oncogene (Bartkova et al, 1996;Ishikawa et al, 2009;Liu et al, 2010;Smith et al, 2007), though they can be stimulated to differentiate through serum deprivation (Croslan et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Integrated analysis of mRNA and microRNA expression in mature neurons, neural progenitor cells and neuroblastoma cells

Liu

Ander

Tian

et al. 2012

Gene

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Mitotic Bypass Via An Occult Cell Cycle Phase Following DNA Topoisomerase II Inhibition In p53 Functional Human Tumor Cells

Cited by 17 publications

References 32 publications

Impact of overexpression of metallothionein-1 on cell cycle progression and zinc toxicity

Impact of overexpression of metallothionein-1 on cell cycle progression and zinc toxicity

Flow-Based Cytometric Analysis of Cell Cycle via Simulated Cell Populations

Integrated analysis of mRNA and microRNA expression in mature neurons, neural progenitor cells and neuroblastoma cells

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