Modeling and optimization of alpha-amylase production in a recombinant yeast fed-batch culture taking account of the cell cycle population distribution.

A simple mathematical model describing the cell cycle dependency of rice alpha-amylase production by a recombinant yeast was constructed to investigate the efficiency of cell cycle population control. First, the effects of the glucose concentration and cultivation temperature on the specific growth rate, the specific production rate of rice alpha-amylase, and the distribution of the cell cycle population were studied under balanced growth conditions. On the basis of the results, parameter values for the mathematical model were then estimated. The proposed model was shown to be applicable for unbalanced as well as balanced growth phases. The optimal control strategy in respect of temperature and glucose concentration for maximum rice alpha-amylase production, taking into account the cell cycle population, was determined and the result was compared with that obtained by a simple mathematical model in which cell cycle distribution was not considered. Finally, the effect of the initial population of each cell cycle phase on the final amount of the product under optimal operational conditions was investigated. The simulation and experimental data coincided well with each other, and the model was used to optimize the control strategy for maximum alpha-amylase production.     

Model-based analysis of Arabidopsis leaf epidermal cells reveals distinct division and expansion patterns for pavement and guard cells

To efficiently capture sunlight for photosynthesis, leaves typically develop into a flat and thin structure. This development is driven by cell division and expansion, but the individual contribution of these processes is currently unknown, mainly because of the experimental difficulties to disentangle them in a developing organ, due to their tight interconnection. To circumvent this problem, we built a mathematic model that describes the possible division patterns and expansion rates for individual epidermal cells. This model was used to fit experimental data on cell numbers and sizes obtained over time intervals of 1 d throughout the development of the first leaf pair of Arabidopsis (Arabidopsis thaliana). The parameters were obtained by a derivative-free optimization method that minimizes the differences between the predicted and experimentally observed cell size distributions. The model allowed us to calculate probabilities for a cell to divide into guard or pavement cells, the maximum size at which it can divide, and its average cell division and expansion rates at each point during the leaf developmental process. Surprisingly, average cell cycle duration remained constant throughout leaf development, whereas no evidence for a maximum cell size threshold for cell division of pavement cells was found. Furthermore, the model predicted that neighboring cells of different sizes within the epidermis expand at distinctly different relative rates, which could be verified by direct observations. We conclude that cell division seems to occur independently from the status of cell expansion, whereas the cell cycle might act as a timer rather than as a size-regulated machinery.     

A computer model of spermatogenesis in the rat; correlation with flow cytometric data based on autoradiographic cell-cycle properties

A computer model of rat spermatogenesis was created, based on autoradiographic studies of durations of the phases of the cell cycle (G1, S, G2 and mitotic phases) of each germ-cell type. With this model it is possible to predict and to gain insight into the changes of the DNA content occurring during the normal process of spermatogenesis. The relative proportions of haploid, diploid, S phase and tetraploid germ cells with increasing age of the rats were calculated. Calculated and actual experimental flow cytometry data were compared to test the accuracy of the model, and these show good agreement. The present work demonstrates that single-parameter DNA analysis of testicular cells is primarily a reflection of germ cells in the spermatocyte and spermatid stages of development, and of non-germ cells. The FCM single-parameter DNA analysis of testicular cells is relatively insensitive to changes in the stem cell and spermatogonial stages of germ-cell development.     

A model describing cell polyploidization in tissues of growing fruit as related to cessation of cell proliferation

Endoreduplication is a phenomenon, widespread among plants, which consists of an incomplete cell cycle without mitosis and leads to the increase of the nuclear DNA content. In this work, a model was developed describing cell proliferation and DNA endoreduplication over the whole fruit development, from the pre-anthesis period until maturation. In each mitotic cycle of duration tau, the proportion of cells proceeding through division depends on a constant parameter rho and on the progressive decline of the proliferating capacity . The non-dividing cells may either stop the reduplication fully, or switch to repeated syntheses of DNA without cell division, resulting in cell endoreduplication. A single constant parameter sigma describes the proportion of cells that moves from one to the next class of DNA content after each lapse of time tauE, considered to be the minimum time required for an endocycle. The model calculates the total number of cells and their distribution among eight classes of ploidy level. The dynamic patterns of cell proliferation and ploidy were compared with those obtained experimentally on two contrasting tomato genotypes. The approach developed in this model should allow the future integration of new knowledge concerning the genetic and environmental control of the switch from complete to incomplete cell cycle.     

Cell population dynamics during the differentiative phase of tissue development

The dynamics of a cell population whose numbers are growing exponentially have been described well by a mathematical model based on the theory of age-dependent branching processes. Such a model, however, does not cover the period following exponential growth when cell differentiation curtails population size. This paper offers an extension to the branching process model to remedy this deficiency. The extended model is ideal for describing embryonic growth; its use is illustrated with data from embryonic retina. The model offers a better computational framework for the interpretation of a variety of data (growth curves of cell numbers, DNA histograms, thymidine labelling indices, FLM curves, BUdR-labelled mitoses curves) because age-distributions can be calculated at any stage of development, not just during exponential growth. Proportions of cells in the various phases of the cell cycle can be computed as growth slows. Such calculations show the gradual transition from a population dominated by cells which are young with respect to cell cycle age to one dominated by those which are old, and the effects such biases have on the proportions of cells in each phase.     

3-O-methylfunicone, a secondary metabolite produced by Penicillium pinophilum, induces growth arrest and apoptosis in HeLa cells

3-O-Methylfunicone (OMF) is a secondary metabolite produced by the soil fungus Penicillium pinophilum which has cytostatic properties. The aim of this study was to investigate the mechanisms by which such properties are exerted, with special reference to any anti-proliferative and apoptotic potential, on HeLa cells. OMF treatment caused about 44% inhibition of cell growth after 24 h, and modifications in the tubulin fibre organization. In addition, a significant increase in p21 mRNA expression and a decrease in cyclin D1 and Cdk4 mRNA expression resulted at the same time. Apoptosis induction was demonstrated by the annexin V assay, cytofluorimetric analysis of the DNA content of the sub-G1 fraction and DNA laddering. Taken together, our data showed that the compound inhibits proliferation of HeLa cells by several mechanisms, such as disruption of tubulin fibres, cell cycle arrest and apoptosis induction. The capacity of the compound to affect the cell cycle and to modulate apoptosis is indicative of a potential for the development of a new agent for cancer chemotherapy.     

Design and synthesis of new antitumor agents with the 1,7-epoxycyclononane framework. Study of their anticancer action mechanism by a model compound

This article describes the design, synthesis and biological evaluation of a new family of antitumor agents having the 1,7-epoxycyclononane framework. We have developed a versatile synthetic methodology that allows the preparation of a chemical library with structural diversity and in good yield. The synthetic methodology has been scaled up to the multigram level and can be developed in an enantioselective fashion. The study in vitro of a model compound, in front of the cancer cell lines HL-60 and MCF-7, showed a growth inhibitory effect better than that of cisplatin. The observation of cancer cells by fluorescence microscopy showed the presence of apoptotic bodies and a degradation of microtubules. The study of cell cycle and mechanism of death of cancer cells by flow cytometry indicates that the cell cycle arrested at the G₀/G₁ phase and that the cells died by apoptosis preferably over necrosis. A high percentage of apoptotic cells at the subG₀/G₁ level was observed. This indicates that our model compound does not behave as an antimitotic agent like nocodazole, used as a reference, which arrests the cell cycle at G₂/M phase. The interaction of anticancer agents with DNA molecules was evaluated by atomic force microscopy, circular dichroism and electrophoresis on agarose gel. The results indicate that the model compound has not DNA as a target molecule. The in silico study of the model compound showed a potential good oral bioavailability.     

Daio-Orengedokudo works as a cell-proliferating compound in endothelial cells

Daio-Orengedokuto is a combination drug that has inhibitory effects on HMG-CoA reductase and pancreatic lipase. Here we investigated whether Daio-Orengedokuto has effects on vascular endothelial cells. To determine its effects on blood vessels, we examined roles of Daio-Orengedokuto in cell migration, cell apoptosis and cell cycle progression over bovine aortic endothelial cells (BAECs). Interestingly, Daio-Orengedokuto was shown to work as an anti-apoptotic agent, a cell cycle progressive agent and a cell-migration inducing agent in BAECs, whereas it was known to act as a tumor suppressor in cancer cells (unpublished data). The inducing effect of Daio-Orengedokuto on cell-cycle progression and cell migration in endothelium suggests that Daio-Orengedokuto may be referred to as a drug, inducing angiogenesis, healing wounds, and (or) remodeling vascular tissue. Then we further investigated which signaling molecules were activated by Daio-Orengedokuto and found that extracellular signal-regulated kinase (ERK) phosphorylation and IkappaB degradation were stimulated by the Daio-Orengedokuto treatment in BAECs. More interestingly, pretreatment with PD compound, an ERK inhibitor, blocked the anti-apoptosis induced by Daio-Orengedokuto. In conclusion, Daio-Orengedokuto plays a role in endothelial cell proliferation via activation of MAP kinase.     

Distribution of SH-SY5Y human neuroblastoma cells in the cell cycle following exposure to organophosphorus compounds

The current study investigated the relationship of the cell cycle phase (as G(0)/G(1), S, and G(2)/M) and cytotoxicity (as sub-G(1) DNA) to determine whether alterations in cell replication were associated with organophosphate (OP) compound induced cytotoxicity. Results demonstrated that, overall, OP compound--induced cell cycle changes were variable and depended on the OP compound, exposure concentration, and temporal relationship to cytotoxicity. Noncytotoxic OP compound treatments substantially decreased the percentage of cells in S phase of the cell cycle when compared to controls. A corresponding increase was seen in the percent of cells in G(0)/G(1) phase of the cell cycle. In the precytotoxic interval of exposure, most cytotoxic OP compound treatments substantially decreased the percentage of cells in G(2)/M phase of the cell cycle. Corresponding increases were seen primarily in G(0)/G(1) phase cells. Effects on cells in S stage of the cell cycle varied with the OP compound. In the during cytotoxic interval of exposure, most cytotoxic OP compound treatments substantially increased the percentage of cells in S phase of the cell cycle. A corresponding decrease in the percent of cells in G(0)/G(1) stage of the cell cycle was observed. Furthermore, treatments either increased or decreased the percentage of cells in G(2)/M phase of the cell cycle when compared to controls, with decreases more likely with the most cytotoxic OP compound exposures. Overall, the in vitro data suggest that exposure to OP compounds can alter the cell cycle status of SH-SY5Y neuroblastoma cells depending on compound, concentration, and interval from initial exposure. Changes in cell cycle, however, did not differentiate between OP compounds that are known for their ability to acutely inhibit acetylcholinesterase versus those inducing type I and type II delayed neurotoxicity.     

Modelling cell population growth with applications to cancer therapy in human tumour cell lines

In this paper we present an overview of the work undertaken to model a population of cells and the effects of cancer therapy. We began with a theoretical one compartment size structured cell population model and investigated its asymptotic steady size distributions (SSDs) (On a cell growth model for plankton, MMB JIMA 21 (2004) 49). However these size distributions are not similar to the DNA (size) distributions obtained experimentally via the flow cytometric analysis of human tumour cell lines (data obtained from the Auckland Cancer Society Research Centre, New Zealand). In our one compartment model, size was a generic term, but in order to obtain realistic steady size distributions we chose size to be DNA content and devised a multi-compartment mathematical model for the cell division cycle where each compartment corresponds to a distinct phase of the cell cycle (J. Math. Biol. 47 (2003) 295). We then incorporated another compartment describing the possible induction of apoptosis (cell death) from mitosis phase (Modelling cell death in human tumour cell lines exposed to anticancer drug paclitaxel, J. Math. Biol. 2004, in press). This enabled us to compare our model to flow cytometric data of a melanoma cell line where the anticancer drug, paclitaxel, had been added. The model gives a dynamic picture of the effects of paclitaxel on the cell cycle. We hope to use the model to describe the effects of other cancer therapies on a number of different cell lines.     

Glyphosate-based pesticides affect cell cycle regulation

Cell-cycle dysregulation is a hallmark of tumor cells and human cancers. Failure in the cell-cycle checkpoints leads to genomic instability and subsequent development of cancers from the initial affected cell. A worldwide used product Roundup 3plus, based on glyphosate as the active herbicide, was suggested to be of human health concern since it induced cell cycle dysfunction as judged from analysis of the first cell division of sea urchin embryos, a recognized model for cell cycle studies. Several glyphosate-based pesticides from different manufacturers were assayed in comparison with Roundup 3plus for their ability to interfere with the cell cycle regulation. All the tested products, Amega, Cargly, Cosmic, and Roundup Biovert induced cell cycle dysfunction. The threshold concentration for induction of cell cycle dysfunction was evaluated for each product and suggests high risk by inhalation for people in the vicinity of the pesticide handling sprayed at 500 to 4000 times higher dose than the cell-cycle adverse concentration.     

Inferring Growth Control Mechanisms in Growing Multi-cellular Spheroids of NSCLC Cells from Spatial-Temporal Image Data

We develop a quantitative single cell-based mathematical model for multi-cellular tumor spheroids (MCTS) of SK-MES-1 cells, a non-small cell lung cancer (NSCLC) cell line, growing under various nutrient conditions: we confront the simulations performed with this model with data on the growth kinetics and spatial labeling patterns for cell proliferation, extracellular matrix (ECM), cell distribution and cell death. We start with a simple model capturing part of the experimental observations. We then show, by performing a sensitivity analysis at each development stage of the model that its complexity needs to be stepwise increased to account for further experimental growth conditions. We thus ultimately arrive at a model that mimics the MCTS growth under multiple conditions to a great extent. Interestingly, the final model, is a minimal model capable of explaining all data simultaneously in the sense, that the number of mechanisms it contains is sufficient to explain the data and missing out any of its mechanisms did not permit fit between all data and the model within physiological parameter ranges. Nevertheless, compared to earlier models it is quite complex i.e., it includes a wide range of mechanisms discussed in biological literature. In this model, the cells lacking oxygen switch from aerobe to anaerobe glycolysis and produce lactate. Too high concentrations of lactate or too low concentrations of ATP promote cell death. Only if the extracellular matrix density overcomes a certain threshold, cells are able to enter the cell cycle. Dying cells produce a diffusive growth inhibitor. Missing out the spatial information would not permit to infer the mechanisms at work. Our findings suggest that this iterative data integration together with intermediate model sensitivity analysis at each model development stage, provide a promising strategy to infer predictive yet minimal (in the above sense) quantitative models of tumor growth, as prospectively of other tissue organization processes. Importantly, calibrating the model with two nutriment-rich growth conditions, the outcome for two nutriment-poor growth conditions could be predicted. As the final model is however quite complex, incorporating many mechanisms, space, time, and stochastic processes, parameter identification is a challenge. This calls for more efficient strategies of imaging and image analysis, as well as of parameter identification in stochastic agent-based simulations.     

A novel model for studying Baculovirus infection process

In this paper, a new Ansatz for modelling the Baculovirus infection cycle is presented. The base of this model is the cell cycle distribution at the time of infection. It is possible to calculate the growth of the culture and the initiation of virus processing by considering cell cycle distribution. By taking into account the length of the viral genome and the polymerase activity, it is possible to calculate the virus production rate, which underlies a logistic growth. In the present work, a new hypothesis explaining the accelerated death rates of infected cells has been introduced. This assumption provides the possibilities of performing calculation without any fixed time intervals. The simulation was tested by comparing experimental data with the model prediction. Therefore, cell cycle distributions over the culture time and the growth behaviour of infected and non-infected insect cells were measured. A model, Baculovirus coding for GFP was employed for the present investigation, as it allows tracking the infection and determining the effectiveness of the infection, which is highly dependent on the cell density at the time of infection (TOI). Furthermore, the new model is is taken to simulate data gained from literature about virus release and adsorption. The new assumptions make the model more independent to fit into different cultivation systems.     

A promising action of riboflavin as a mediator of leukaemia cell death

Besides having a pivotal biological function as a component of coenzymes, riboflavin appears a promissing antitumoral agent, but the underlying molecular mechanism remains unclear. In this work, we demonstrate that irradiated riboflavin, when applied at μM concentrations, induces an orderly sequence of signaling events finally leading to leukemia cell death. The molecular mechanism involved is dependent on the activation of caspase 8 caused by overexpression of Fas and FasL and also on mitochondrial amplification mechanisms, involving the stimulation of ceramide production by sphingomyelinase and ceramide synthase. The activation of this cascade led to an inhibition of mitogen activated protein kinases: JNK, MEK and ERK and survival mediators (PKB and IAP1), upregulation of the proapoptotic Bcl2 member Bax and downregulation of cell cycle progression regulators. Importantly, induction of apoptosis by irradiated riboflavin was leukaemia cell specific, as normal human lymphocytes did not respond to the compound with cell death. Our data indicate that riboflavin selectively activates Fas cascade and also constitutes a death receptor-engaged drug without harmful side effects in normal cells, bolstering the case for using this compound as a novel avenue for combating cancerous disease.     

Quantifying the Length and Variance of the Eukaryotic Cell Cycle Phases by a Stochastic Model and Dual Nucleoside Pulse Labelling

A fundamental property of cell populations is their growth rate as well as the time needed for cell division and its variance. The eukaryotic cell cycle progresses in an ordered sequence through the phases and and is regulated by environmental cues and by intracellular checkpoints. Reflecting this regulatory complexity, the length of each phase varies considerably in different kinds of cells but also among genetically and morphologically indistinguishable cells. This article addresses the question of how to describe and quantify the mean and variance of the cell cycle phase lengths. A phase-resolved cell cycle model is introduced assuming that phase completion times are distributed as delayed exponential functions, capturing the observations that each realization of a cycle phase is variable in length and requires a minimal time. In this model, the total cell cycle length is distributed as a delayed hypoexponential function that closely reproduces empirical distributions. Analytic solutions are derived for the proportions of cells in each cycle phase in a population growing under balanced growth and under specific non-stationary conditions. These solutions are then adapted to describe conventional cell cycle kinetic assays based on pulse labelling with nucleoside analogs. The model fits well to data obtained with two distinct proliferating cell lines labelled with a single bromodeoxiuridine pulse. However, whereas mean lengths are precisely estimated for all phases, the respective variances remain uncertain. To overcome this limitation, a redesigned experimental protocol is derived and validated in silico. The novelty is the timing of two consecutive pulses with distinct nucleosides that enables accurate and precise estimation of both the mean and the variance of the length of all phases. The proposed methodology to quantify the phase length distributions gives results potentially equivalent to those obtained with modern phase-specific biosensor-based fluorescent imaging.     

Monocytic differentiation of HL60 cells induced by potent analogs of vitamin D3 precedes the G1/G0 phase cell cycle block

Abstract. Differentiation of mammalian cells is accompanied by reduced rates of proliferation and an exit from the cell cycle. Human leukemic cells HL60 present a widely used model of neoplastic cell differentiation, and acquire the monocytic phenotype when exposed to analogs of vitamin D₃ (VD₃). The maturation process is accompanied by two blocks in the cell cycle: an arrest in the G₁/G₀ phase, and a recently described G₂+ M block. In this study we have analyzed the traverse of the cell cycle phases of the well-differentiating HL60-G cells exposed to one of ten analogs of VD₃, and compared the cell cycle effects of each compound with its potency as a differentiation-inducing agent. We found that in general there was a good correlation between the effects of these compounds on the cell cycle and on differentiation, but the best cell cycle predictor of differentiation potency was the extent of accumulation of the cells in the G₂ compartment. All analogs induced a marked decrease in the mitotic index, and polynucleation of HL60 cells was produced, especially by compounds which were effective as inducers of differentiation. Time course studies showed that induction of differentiation was accompanied by a transient increase of the proportion of cells in the G₂+ M compartment, but preceded the G₁ to S, and the G₂ compartment blocks. These studies indicate that complex changes in the cell cycle traverse accompany, but do not precede, the acquisition of the monocytic phenotype by HL60 cells.