Ab initio phenomenological simulation of the growth of large tumor cell populations

In a previous paper we have introduced a phenomenological model of cell metabolism and of the cell cycle to simulate the behavior of large tumor cell populations (Chignola and Milotti 2005 Phys. Biol. 2 8). Here we describe a refined and extended version of the model that includes some of the complex interactions between cells and their surrounding environment. The present version takes into consideration several additional energy-consuming biochemical pathways such as protein and DNA synthesis, the tuning of extracellular pH and of the cell membrane potential. The control of the cell cycle, which was previously modeled by means of ad hoc thresholds, has been directly addressed here by considering checkpoints from proteins that act as targets for phosphorylation on multiple sites. As simulated cells grow, they can now modify the chemical composition of the surrounding environment which in turn acts as a feedback mechanism to tune cell metabolism and hence cell proliferation: in this way we obtain growth curves that match quite well those observed in vitro with human leukemia cell lines. The model is strongly constrained and returns results that can be directly compared with actual experiments, because it uses parameter values in narrow ranges estimated from experimental data, and in perspective we hope to utilize it to develop in silico studies of the growth of very large tumor cell populations (10(6) cells or more) and to support experimental research. In particular, the program is used here to make predictions on the behavior of cells grown in a glucose-poor medium: these predictions are confirmed by experimental observation.     

Hemichannel-Mediated and pH-Based Feedback from Horizontal Cells to Cones in the Vertebrate Retina

Background

Recent studies designed to identify the mechanism by which retinal horizontal cells communicate with cones have implicated two processes. According to one account, horizontal cell hyperpolarization induces an increase in pH within the synaptic cleft that activates the calcium current (Ca²⁺-current) in cones, enhancing transmitter release. An alternative account suggests that horizontal cell hyperpolarization increases the Ca²⁺-current to promote transmitter release through a hemichannel-mediated ephaptic mechanism.

Methodology/Principal Findings

To distinguish between these mechanisms, we interfered with the pH regulating systems in the retina and studied the effects on the feedback responses of cones and horizontal cells. We found that the pH buffers HEPES and Tris partially inhibit feedback responses in cones and horizontal cells and lead to intracellular acidification of neurons. Application of 25 mM acetate, which does not change the extracellular pH buffer capacity, does lead to both intracellular acidification and inhibition of feedback. Because intracellular acidification is known to inhibit hemichannels, the key experiment used to test the pH hypothesis, i.e. increasing the extracellular pH buffer capacity, does not discriminate between a pH-based feedback system and a hemichannel-mediated feedback system. To test the pH hypothesis in a manner independent of artificial pH-buffer systems, we studied the effect of interfering with the endogenous pH buffer, the bicarbonate/carbonic anhydrase system. Inhibition of carbonic anhydrase allowed for large changes in pH in the synaptic cleft of bipolar cell terminals and cone terminals, but the predicted enhancement of the cone feedback responses, according to the pH-hypothesis, was not observed. These experiments thus failed to support a proton mediated feedback mechanism. The alternative hypothesis, the hemichannel-mediated ephaptic feedback mechanism, was therefore studied experimentally, and its feasibility was buttressed by means of a quantitative computer model of the cone/horizontal cell synapse.

Conclusion

We conclude that the data presented in this paper offers further support for physiologically relevant ephaptic interactions in the retina.     

Effects of organic pH buffers on a cell growth and an antibody production of human-human hybridoma HB4C5 cells in a serum-free culture

Human-human hybridoma cells secreting a human monoclonal antibody were cultured in a serum-free medium containing various organic pH buffers in order to clarify their effects on cell growth and antibody production. Organic pH buffers having either one sulfonic acid and several acyclic amine moieties, or several cyclic amine moieties containing two amino nitrogen did not inhibit cell growth; while other organic buffers sulfonic acid moiety plus several cyclic amine moieties containing one amino nitrogen slightly decreased cell growth, but enhanced antibody production. Using Fujita's organic conceptual diagram, a relationship between the organicity and inorganicity of a pH buffer to cell growth and antibody production was found. pH buffers with large inorganicity and small organicity values were favorable for cell growth, and buffers with small inorganicity and large organicity values were preferred to enhance antibody production. Although the pH buffering range affects cell growth, its effect on antibody production is not clear. In conclusion, 2-morpholinoethanesulfonic acid (MES), 3-morpholino-propanesulfonic acid (MOPS) and 1, 2-N, N-bis[N, N-di(2-sulfonoethyl)piperazinyl]ethane (Bis-PIPES) are shown to be the most optimal of the buffers tested, because they enhanced antibody production without decreasing the cell growth among the pH buffers tested here.     

L1CAM ubiquitination facilitates its lysosomal degradation

The cell adhesion molecule L1 is implicated in several processes in the developing and adult nervous system. Intracellular trafficking of L1 is important for cell migration, neurite growth and adhesion. We demonstrate here that L1 is ubiquitinated at the plasma membrane and in early endosomes. Mono-ubiquitination regulates L1 intracellular trafficking by enhancing its lysosomal degradation. We propose that L1’s ubiquitination might be an additional mechanism to control its re-appearance at the cell surface thereby influencing processes like neurite growth and cell adhesion.     

Feeder cells impart a growth stimulus to recipient epithelial cells by direct contact.

Mouse mammary epithelial cells have been shown to proliferate when cultured in the same vessel with lethally irradiated cells of the LA7 rat mammary tumor line. Presented here are experiments that indicate that the LA7 feeder cells stimulate growth of the normal mouse mammary cells by a mechanism that involves direct contact between the two cell types. It is possible that the LA7 feeder cells stimulate proliferation by secretion of a labile growth factor, by secretion of a soluble growth factor in such low concentrations that dilution by travel over a distance makes it less effective, that the stimulus is transduced directly through membrane receptors on the recipient epithelial cells, or that a growth message is sent through gap junctions between cells. This feeder cell system is proposed as an in vitro model for epithelial wound healing.     

Sex hormone-binding globulin (SHBG) and estradiol cross-talk in breast cancer cells.

Sex hormone-binding globulin (SHBG) is a plasma glycoprotein that regulates the action of steroid hormones at several levels. SHBG regulates the availability of free androgens and estradiol to hormone-responsive tissues. Moreover, SHBG is also part of a novel steroid signaling system. We report here on the mechanism of action and the biological effects of SHBG in breast cancer cells, especially distinguishing cross-talk between membrane-initiated SHBG and estradiol pathways. After interacting with a specific binding site on breast cancer cell membranes, SHBG activates a specific pathway, and by cAMP induction, inhibits estradiol-mediated activation of ERK. Both estradiol and SHBG membrane-initiated pathways involve cross-talk at MAP kinase level with the ultimate result of inhibiting estradiol-mediated cell growth and antiapoptosis. On the basis of reported evidence, we suggest that SHBG is one of the regulators of growth and apoptosis of estrogen-dependent breast cancer cells.     

Linking cell division to cell growth in a spatiotemporal model of the cell cycle

Cell division must be tightly coupled to cell growth in order to maintain cell size, yet the mechanisms linking these two processes are unclear. It is known that almost all proteins involved in cell division shuttle between cytoplasm and nucleus during the cell cycle; however, the implications of this process for cell cycle dynamics and its coupling to cell growth remains to be elucidated. We developed mathematical models of the cell cycle which incorporate protein translocation between cytoplasm and nucleus. We show that protein translocation between cytoplasm and nucleus not only modulates temporal cell cycle dynamics, but also provides a natural mechanism coupling cell division to cell growth. This coupling is mediated by the effect of cytoplasmic-to-nuclear size ratio on the activation threshold of critical cell cycle proteins, leading to the size-sensing checkpoint (sizer) and the size-independent clock (timer) observed in many cell cycle experiments.     

The C. elegans hmr-1 gene can encode a neuronal classic cadherin involved in the regulation of axon fasciculation.

Nervous system morphogenesis is characterized by extensive interactions between individual axon growth cones and their cellular environments. Selective cell adhesion is one mechanism by which the growth of an axon can be modulated, and members of the classic cadherin group of cell adhesion molecules have been shown to play a role in this process in both vertebrates and Drosophila. In Drosophila, there are two classic cadherins: one involved primarily in regulating the morphogenesis of epithelia, and the other, DN-cadherin, required almost exclusively in neuronal development. In contrast, C. elegans has a single classic cadherin gene, hmr-1, whose function is required for epithelial morphogenesis. We show here that hmr-1 also encodes a second classic cadherin via a novel mechanism involving an alternative, neuron-specific promoter, coupled with alternative splicing. This novel HMR-1 isoform is very similar to DN-cadherin, and a mutant strain that specifically lacks the function of this isoform displays defects in the fasciculation and outgrowth of a subset of motor neuron processes; a phenotype that resembles loss of DN-cadherin function in Drosophila. These results indicate that Drosophila and C. elegans share a conserved, cadherin-dependent mechanism involved in regulating axonal patterning and fasciculation.     

Translation of CUG- but not AUG-initiated forms of human fibroblast growth factor 2 is activated in transformed and stressed cells

Four isoforms of the human fibroblast growth factor 2 (FGF-2), with different intracellular localizations and distinct effects on cell phenotype, result from alternative initiations of translation at three CUG and one AUG start codons. We showed here by Western immunoblotting and immunoprecipitation that the CUG-initiated forms of FGF-2 were synthesized in transformed cells, whereas "normal" cells almost exclusively produced the AUG-initiated form. CUG-initiated FGF-2 was induced in primary skin fibroblasts in response to heat shock and oxidative stress. In transformed cells and in stressed fibroblasts, CUG expression was dependent on cis-elements within the 5'' region of FGF-2 mRNA and was not correlated to mRNA level, indicating a translational regulation. UV cross-linking experiments revealed that CUG expression was linked to the binding of several cellular proteins to FGF-2 mRNA 5'' region. Since translation of FGF-2 mRNA was previously shown to occur by internal ribosome entry, a nonclassical mechanism already described for picornaviruses, the cross-linking patterns of FGF-2 and picornavirus mRNAs were compared. Comigration of several proteins, including a p60, was observed. However, this p60 was shown to be different from the p57/PTB internal entry factor, suggesting a specificity towards FGF-2 mRNA. We report here a process of translational activation of the FGF-2 CUG-initiated forms in direct relation with trans-acting factors specific to transformed and stressed cells. These data favor a critical role of CUG-initiated FGF-2 in cell transformation and in the stress response.     

Small-scale bioreactor system for process development and optimization

An agitated 12-well microtiter plate system with a working volume of 2ml was investigated for cell culture process development. Agitation assures homogeneity in wells and enhances mass transfer between the gas and the liquid phase, thus improving maximum cell density and pH stability. The pH of the NaHCO(3)-buffered system can be adjusted by altering the carbon dioxide content of the gas phase. The non-toxic, visual pH indicator phenol red was used in combination with a spectrophotometric plate reader for rapid and precise pH measurements. For high throughputs, cell growth was assessed non-invasively using stable green fluorescent protein (GFP) expressing cells and a fluorescence plate reader. The setup is simple and inexpensive. The system can be automated and allows several hundred small-scale bioreactor experiments to be run in parallel.     

Impaired growth in transgenic plants over-expressing an expansin isoform

Expansins are cell wall proteins characterised by their ability to stimulate wall loosening during cell expansion. The expression of some expansin isoforms is clearly correlated with growth and the external application of expansins can stimulate cell expansion in vivo in several systems. We report here the expression of a heterologous expansin coding sequence in transgenic tomato plants (Lycopersicon esculentum Mill.) under the control of a constitutive promoter. In some transgenic lines with high levels of expansin activity extractable from cell walls, we observed alterations of growth: mature plants were stunted, with shorter leaves and internodes, and dark-grown seedlings had shorter and wider hypocotyls than their wild-type counterparts. Examination of hypocotyl sections revealed similar differences at the cellular level: cortical and epidermal cells were shorter and wider than those from wild-type seedlings. The observed stimulation of radial expansion did not compensate for the decreased elongation, and overall growth was reduced in the transgenics. As this observation can seem paradoxical given the known effect of expansins on isolated cell walls, we examined the mechanical behaviour of transgenic tissue. We measured a decrease in hypocotyl elongation in response to acidic pH in the transformants. This result may account for the alterations in cell expansion, and could itself be explained by a reduced susceptibility of transgenic cell walls to expansin action.     

Role of neutrophil extracellular traps in regulation of lung cancer invasion and metastasis: Structural insights from a computational model

Lung cancer is one of the leading causes of cancer-related deaths worldwide and is characterized by hijacking immune system for active growth and aggressive metastasis. Neutrophils, which in their original form should establish immune activities to the tumor as a first line of defense, are undermined by tumor cells to promote tumor invasion in several ways. In this study, we investigate the mutual interactions between the tumor cells and the neutrophils that facilitate tumor invasion by developing a mathematical model that involves taxis-reaction-diffusion equations for the critical components in the interaction. These include the densities of tumor and neutrophils, and the concentrations of signaling molecules and structure such as neutrophil extracellular traps (NETs). We apply the mathematical model to a Boyden invasion assay used in the experiments to demonstrate that the tumor-associated neutrophils can enhance tumor cell invasion by secreting the neutrophil elastase. We show that the model can both reproduce the major experimental observation on NET-mediated cancer invasion and make several important predictions to guide future experiments with the goal of the development of new anti-tumor strategies. Moreover, using this model, we investigate the fundamental mechanism of NET-mediated invasion of cancer cells and the impact of internal and external heterogeneity on the migration patterning of tumour cells and their response to different treatment schedules.     

Growth factors and apoptosis

Abstract. Apoptosis is nowadays recognized as an important mechanism by which cells can be eliminated from the organism. In particular its role in tissue modelling during embryogenesis has been highlighted. The human teratoma cell line Tera 2, which in several respects acts as a human embryonic stem cell, can be induced to undergo apoptosis by reducing the serum content of the tissue culture medium. We report here that this process can be reversed by replacing serum with the heparin-binding growth factors, acidic FGF and basic FGF. In contrast, neither of the mammalian transforming growth factors (TGF-β-3) managed to exert any effect on growth or apoptosis in Tera 2 cells.     

Dynamics and modeling on fermentative production of poly (β-hydroxybutyric acid) from sugars via lactate by a mixed culture of Lactobacillus delbrueckii and Alcaligenes eutrophus

The mixed culture system was considered in the present research where sugars such as glucose were converted to lactate by Lactobacillus delbrueckii and the lactate was converted to poly β-hydroxybutyrate (PHB) by Alcaligenes eutrophus in one fermentor. For the modeling of the effect of NH₃ concentration on the cell growth of A. eutrophus and PHB production rates, metabolic flux distributions were computed at two culture phases of cell growth and PHB production periods. It was found that the NADPH, generated through isocitrate dehydrogenate in TCA cycle, was predominantly utilized for the reaction from α-ketoglutalate to glutamate when NH₃ was abundant, while it tended to be utilized for the PHB production through acetoacetyl CoA reductase as NH₃ concentration decreased. This phenomenon was reflected in the development of mathematical model. In the mixed culture experiments, the two phases were observed, namely the lactate production phase due to L. delbrueckii and the lactate consumption phase due to A. eutrophus. The lactate concentration could be estimated on-line by the amount of NaOH solution and HCl solution supplied to keep the culture pH at constant level. Several mixed culture experiments were conducted to see the dynamics of the system. Finally, a mathematical model which can describe the dynamic behavior of the present mixed culture was developed and the model parameters were tuned for fitting the experimental data. The model may be used for several purposes such as control, optimization, and understanding process dynamics etc.     

Persistent Effectivity of Gas Plasma-Treated,Long Time-Stored Liquid on Epithelial Cell Adhesion Capacity and Membrane Morphology

Research in plasma medicine includes a major interest in understanding gas plasma-cell interactions. The immediate application of gas plasma in vitro inhibits cell attachment, vitality and cell-cell contacts via the liquid. Interestingly, in our novel experiments described here we found that the liquid-mediated plasma effect is long-lasting after storage up to seven days; i. e. the liquid preserves the characteristics once induced by the argon plasma. Therefore, the complete Dulbecco''s Modified Eagle cell culture medium was argon plasma-treated (atmospheric pressure, kINPen09) for 60 s, stored for several days (1, 4 and 7 d) at 37°C and added to a confluent mouse hepatocyte epithelial cell (mHepR1) monolayer. Impaired tight junction architecture as well as shortened microvilli on the cell membrane could be observed, which was accompanied by the loss of cell adhesion capacity. Online-monitoring of vital cells revealed a reduced cell respiration. Our first time-dependent analysis of plasma-treated medium revealed that temperature, hydrogen peroxide production, pH and oxygen content can be excluded as initiators of cell physiological and morphological changes. The here observed persisting biological effects in plasma-treated liquids could open new medical applications in dentistry and orthopaedics.     

Aluminum Toxicity in Roots : Correlation among Ionic Currents, Ion Fluxes, and Root Elongation in Aluminum-Sensitive and Aluminum-Tolerant Wheat Cultivars

The inhibition of root growth by aluminum (Al) is well established, yet a unifying mechanism for Al toxicity remains unclear. The association between cell growth and endogenously generated ionic currents measured in many different systems, including plant roots, suggests that these currents may be directing growth. A vibrating voltage microelectrode system was used to measure the net ionic currents at the apex of wheat (Triticum aestivum L.) roots from Al-tolerant and Al-sensitive cultivars. We examined the relationship between these currents and Al-induced inhibition of root growth. In the Al-sensitive cultivar, Scout 66, 10 micromolar Al (pH 4.5) began to inhibit the net current and root elongation within 1 to 3 hours. These changes occurred concurrently in 75% of experiments. A significant correlation was found between current magnitude and the rate of root growth when data were pooled. No changes in either current magnitude or growth rate were observed in similar experiments using the Al-tolerant cultivar Atlas 66. Measurements with ion-selective microelectrodes suggested that H⁺ influx was responsible for most of the current at the apex, with smaller contributions from Ca²⁺ and Cl⁻ fluxes. In 50% of experiments, Al began to inhibit the net H⁺ influx in Scott 66 roots at the same time that growth was affected. However, in more than 25% of cases, Al-induced inhibition of growth rate occurred before any sustained decrease in the current or H⁺ flux. Although showing a correlation between growth and current or H⁺ fluxes, these data do not suggest a mechanistic association between these processes. We conclude that the inhibition of root growth by Al is not caused by the reduction in current or H⁺ influx at the root apex.     

Linking stem cell function and growth pattern of intestinal organoids

Intestinal stem cells (ISCs) require well-defined signals from their environment in order to carry out their specific functions. Most of these signals are provided by neighboring cells that form a stem cell niche, whose shape and cellular composition self-organize. Major features of this self-organization can be studied in ISC-derived organoid culture. In this system, manipulation of essential pathways of stem cell maintenance and differentiation results in well-described growth phenotypes.We here provide an individual cell-based model of intestinal organoids that enables a mechanistic explanation of the observed growth phenotypes. In simulation studies of the 3D structure of expanding organoids, we investigate interdependences between Wnt- and Notch-signaling which control the shape of the stem cell niche and, thus, the growth pattern of the organoids. Similar to in vitro experiments, changes of pathway activities alter the cellular composition of the organoids and, thereby, affect their shape. Exogenous Wnt enforces transitions from branched into a cyst-like growth pattern; known to occur spontaneously during long term organoid expansion. Based on our simulation results, we predict that the cyst-like pattern is associated with biomechanical changes of the cells which assign them a growth advantage. The results suggest ongoing stem cell adaptation to in vitro conditions during long term expansion by stabilizing Wnt-activity.Our study exemplifies the potential of individual cell-based modeling in unraveling links between molecular stem cell regulation and 3D growth of tissues. This kind of modeling combines experimental results in the fields of stem cell biology and cell biomechanics constituting a prerequisite for a better understanding of tissue regeneration as well as developmental processes.     

Modeling the Dynamics of Cdc42 Oscillation in Fission Yeast

Regulation of polarized cell growth is essential for many cellular processes, including spatial coordination of cell morphology changes during growth and division. We present a mathematical model of the core mechanism responsible for the regulation of polarized growth dynamics by the small GTPase Cdc42. The model is based on the competition of growth zones of Cdc42 localized at the cell tips for a common substrate (inactive Cdc42) that diffuses in the cytosol. We consider several potential ways of implementing negative feedback between Cd42 and its GEF in this model that would be consistent with the observed oscillations of Cdc42 in fission yeast. We analyze the bifurcations in this model as the cell length increases, and total amount of Cdc42 and GEF increase. Symmetric antiphase oscillations at two tips emerge via saddle-homoclinic bifurcations or Hopf bifurcations. We find that a stable oscillation and a stable steady state can coexist, which is consistent with the experimental finding that only 50% of bipolar cells oscillate. The mean amplitude and period can be tuned by parameters involved in the negative feedback. We link modifications in the parameters of the model to observed mutant phenotypes. Our model suggests that negative feedback is more likely to be acting through inhibition of GEF association rather than upregulation of GEF dissociation.