Cellular Growth Rate Research Articles

Fibulin 2, a Tyrosine O-Sulfated Protein, Is Up-regulated Following Retinal Detachment

Retinal detachment is the physical separation of the retina from the retinal pigment epithelium. It occurs during aging, trauma, or during a variety of retinal disorders such as age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, or as a complication following cataract surgery. This report investigates the role of fibulin 2, an extracellular component, in retinal detachment. A major mechanism for detachment resolution is enhancement of cellular adhesion between the retina and the retinal pigment epithelium and prevention of its cellular migration. This report shows that fibulin 2 is mainly present in the retinal pigment epithelium, Bruch membrane, choriocapillary, and to a lesser degree in the retina. In vitro studies revealed the presence of two isoforms for fibulin 2. The small isoform is located inside the cell, and the large isoform is present inside and outside the cells. Furthermore, fibulin 2 is post-translationally modified by tyrosine sulfation, and the sulfated isoform is present outside the cell, whereas the unsulfated pool is internally located. Interestingly, sulfated fibulin 2 significantly reduced the rate of cellular growth and migration. Finally, levels of fibulin 2 dramatically increased in the retinal pigment epithelium following retinal detachment, suggesting a direct role for fibulin 2 in the re-attachment of the retina to the retinal pigment epithelium. Understanding the role of fibulin 2 in enhancing retinal attachment is likely to help improve the current therapies or allow the development of new strategies for the treatment of this sight-threatening condition.

Biodegradable polymeric nanoparticles show high efficacy and specificity at DNA delivery to human glioblastoma in vitro and in vivo.

Current glioblastoma therapies are insufficient to prevent tumor recurrence and eventual death. Here, we describe a method to treat malignant glioma by nonviral DNA delivery using biodegradable poly(β-amino ester)s (PBAEs), with a focus on the brain tumor initiating cells (BTICs), the tumor cell population believed to be responsible for the formation of new tumors and resistance to many conventional therapies. We show transfection efficacy of >60% and low biomaterial-mediated cytotoxicity in primary human BTICs in vitro even when the BTICs are grown as 3-D oncospheres. Intriguingly, we find that these polymeric nanoparticles show intrinsic specificity for nonviral transfection of primary human BTICs over primary healthy human neural progenitor cells and that this specificity is not due to differences in cellular growth rate or total cellular uptake of nanoparticles. Moreover, we demonstrate that biodegradable PBAE/DNA nanoparticles can be fabricated, lyophilized, and then stored for at least 2 years without losing efficacy, increasing the translational relevance of this technology. Using lyophilized nanoparticles, we show transgene expression by tumor cells after intratumoral injection into an orthotopic murine model of human glioblastoma. PBAE/DNA nanoparticles were more effective than naked DNA at exogenous gene expression in vivo, and tumor cells were transfected more effectively than noninvaded brain parenchyma in vivo. This work shows the potential of nonviral gene delivery tools to target human brain tumors.

Selenium status affects selenoprotein expression, reproduction, and F1 generation locomotor activity in zebrafish (Danio rerio)

Se is an essential trace element, and is incorporated into selenoproteins which play important roles in human health. Mammalian selenoprotein-coding genes are often present as paralogues in teleost fish, and it is unclear whether the expression patterns or functions of these fish paralogues reflect their mammalian orthologues. Using the model species zebrafish (Danio rerio; ZF), we aimed to assess how dietary Se affects key parameters in Se metabolism and utilisation including glutathione peroxidase (GPX) activity, the mRNA expression of key Se-dependent proteins (gpx1a, gpx1b, sepp1a and sepp1b), oxidative status, reproductive success and F1 generation locomotor activity. From 27 d until 254 d post-fertilisation, ZF were fed diets with graded levels of Se ranging from deficient ( < 0·10 mg/kg) to toxic (30 mg/kg). The mRNA expression of gpx1a and gpx1b and GPX activity responded in a similar manner to changes in Se status. GPX activity and mRNA levels were lowest when dietary Se levels (0·3 mg/kg) resulted in the maximum growth of ZF, and a proposed bimodal mechanism in response to Se status below and above this dietary Se level was identified. The expression of the sepp1 paralogues differed, with only sepp1a responding to Se status. High dietary Se supplementation (30 mg/kg) decreased reproductive success, while the offspring of ZF fed above 0·3 mg Se/kg diet had lower locomotor activity than the other groups. Overall, the novel finding of low selenoprotein expression and activity coinciding with maximum body growth suggests that even small Se-induced variations in redox status may influence cellular growth rates.

Influence of temperature and CO&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt; on the strontium and magnesium composition of coccolithophore calcite

Abstract. Marine calcareous sediments provide a fundamental basis for palaeoceanographic studies aiming to reconstruct past oceanic conditions and understand key biogeochemical element cycles. Calcifying unicellular phytoplankton (coccolithophores) are a major contributor to both carbon and calcium cycling by photosynthesis and the production of calcite (coccoliths) in the euphotic zone, and the subsequent long-term deposition and burial into marine sediments. Here we present data from controlled laboratory experiments on four coccolithophore species and elucidate the relation between the divalent cation (Sr, Mg and Ca) partitioning in coccoliths and cellular physiology (growth, calcification and photosynthesis). Coccolithophores were cultured under different seawater temperature and carbonate chemistry conditions. The partition coefficient of strontium (DSr) was positively correlated with both carbon dioxide (pCO2) and temperature but displayed no coherent relation to particulate organic and inorganic carbon production rates. Furthermore, DSr correlated positively with cellular growth rates when driven by temperature but no correlation was present when changes in growth rates were pCO2-induced. Our results demonstrate the complex interaction between environmental forcing and physiological control on the strontium partitioning in coccolithophore calcite and challenge interpretations of the coccolith Sr / Ca ratio from high-pCO2 environments (e.g. Palaeocene–Eocene thermal maximum). The partition coefficient of magnesium (DMg) displayed species-specific differences and elevated values under nutrient limitation. No conclusive correlation between coccolith DMg and temperature was observed but pCO2 induced a rising trend in coccolith DMg. Interestingly, the best correlation was found between coccolith DMg and chlorophyll a production, suggesting that chlorophyll a and calcite associated Mg originate from the same intracellular pool. These and previous findings indicate that Mg is transported into the cell and to the site of calcification via different pathways than Ca and Sr. Consequently, the coccolith Mg / Ca ratio should be decoupled from the seawater Mg / Ca ratio. This study gives an extended insight into the driving factors influencing the coccolith Mg / Ca ratio and should be considered for future palaeoproxy calibrations.

Metabolic states with maximal specific rate carry flux through an elementary flux mode

Specific product formation rates and cellular growth rates are important maximization targets in biotechnology and microbial evolution. Maximization of a specific rate (i.e. a rate expressed per unit biomass amount) requires the expression of particular metabolic pathways at optimal enzyme concentrations. In contrast to the prediction of maximal product yields, any prediction of optimal specific rates at the genome scale is currently computationally intractable, even if the kinetic properties of all enzymes are available. In the present study, we characterize maximal-specific-rate states of metabolic networks of arbitrary size and complexity, including genome-scale kinetic models. We report that optimal states are elementary flux modes, which are minimal metabolic networks operating at a thermodynamically-feasible steady state with one independent flux. Remarkably, elementary flux modes rely only on reaction stoichiometry, yet they function as the optimal states of mathematical models incorporating enzyme kinetics. Our results pave the way for the optimization of genome-scale kinetic models because they offer huge simplifications to overcome the concomitant computational problems.

Isolation and Characterization of Gasoline-Degrading Yeasts from Refined Oil-Contaminated Residues

Hydrocarbon-degrading yeasts were isolated from three sites contaminated with products of refined petroleum originating from an oily residue treatment Brazilian company. A selective enrichment technique was used by supplementing medium with gasoline, which resulted in eleven isolates; two identified as Rhodotorula mucilaginosa, one as Rhodosporidium diobovatum, four as Meyerozyma (Pichia) guilliermondii, two as Wickerhamia sp., and two as Meyerozyma sp. The strains were evaluated for their growth capacity in medium containing 1% (v/v) gasoline, kerosene or lubricating oil as the only carbon source; the largest values for cellular biomass and growth rates (μ) were observed with gasoline supplementation. The strains were tolerant to aromatic (toluene and xylene) and aliphatic (hexene and n-heptane) compounds, which are part of the composition of gasoline, at concentrations up to 30 mM toluene (0.3% v/v), 20 mM xylene (0.25% v/v), 80 mM n-heptane (1.17% v/v) and 100 mM hexane (1.33% v/v). The R. mucilaginosa S47 and Meyerozyma sp. SP1 strains showed the greatest degradation percentages of gasoline, and have the potential to be used in the bioremediation of gasoline-contaminated environments.

Growth feedback as a basis for persister bistability.

A small fraction of cells in many bacterial populations, called persisters, are much less sensitive to antibiotic treatment than the majority. Persisters are in a dormant metabolic state, even while remaining genetically identical to the actively growing cells. Toxin and antitoxin modules in bacteria are believed to be one possible cause of persistence. A two-gene operon, HipBA, is one of many chromosomally encoded toxin and antitoxin modules in Escherichia coli and the HipA7 allelic variant was the first validated high-persistence mutant. Here, we present a stochastic model that can generate bistability of the HipBA system, via the reciprocal coupling of free HipA to the cellular growth rate. The actively growing state and the dormant state each correspond to a stable state of this model. Fluctuations enable transitions from one to the other. This model is fully in agreement with experimental data obtained with synthetic promoter constructs.

Stability and Oscillation Phenomena Analysis in Microbial Continuous Fermentation Time-Delay System

Based on 1,3-propanediol production from continuous fermentation of glycerol by Klebsiellapneumoniae, the dynamic behaviors of a kinetics system with continuous time-delay are analyzed. At first, a six-dimension kinetics system concerning the intracellular substance is proposed by introducing weak nuclear continuous time-delay into the specific cellular growth rate. Second, taking the inverse of average time-delay as parameter, the local stability of the positive equilibriums and the existence of Hopf bifurcation are discussed. Finally, the period solution of biomass and the parameter region of Hopf bifurcation are pictured numerically taking some special number in substrate concentration in feed medium and dilution rate.

Growth rate-coordinated transcriptome reorganization in bacteria

BackgroundCell growth rate reflects an organism’s physiological state and largely relies on the ability of gene expression to respond to the environment. The relationship between cellular growth rate and gene expression remains unknown.ResultsGrowth rate-coordinated changes in gene expression were discovered by analyzing exponentially growing Escherichia coli cells cultured under multiple defined environments, in which osmotic pressure, temperature and starvation status were varied. Gene expression analyses showed that all 3,740 genes in the genome could be simply divided into three clusters (C1, C2 and C3), which were accompanied by a generic trend in the growth rate that was coordinated with transcriptional changes. The direction of transcriptional change in C1 indicated environmental specificity, whereas those in C2 and C3 were correlated negatively and positively with growth rates, respectively. The three clusters exhibited differentiated gene functions and gene regulation task division.ConclusionsWe identified three gene clusters, exhibiting differential gene functions and distinct directions in their correlations with growth rates. Reverses in the direction of the growth rate correlated transcriptional changes and the distinguished duties of the three clusters indicated how transcriptome homeostasis is maintained to balance the total expression cost for sustaining life in new habitats.

Understanding translational control mechanisms of the mTOR pathway in CHO cells by polysome profiling

The mammalian target of rapamycin (mTOR) pathway plays essential roles in the regulation of translational activity in many eukaryotes. Thus, from a bioprocessing point of view, understanding its molecular mechanisms may provide potential avenues for improving cell culture performance. Toward this end, the mTOR pathway of CHO cells in batch cultures was subjected to rapamycin treatment (inhibition) or nutrient supplementation (induction) and translational activities of CHO cells producing a monoclonal antibody (mAb) were evaluated with polysome profiling technology. Expectedly, rapamycin induced a shift of mRNAs from polysomes towards monosomes, thus reducing maximum cellular growth rate by 30%, while feeding additional nutrients extended mTOR pathway activity during the stationary growth phase in control batch culture, thereby contributing to an increase in global translation activity by up to 2-fold, and up to 5-fold higher specific translation of the heavy and light chains of the recombinant mAb. These increases in translation activity correlated with a 5-day extension in cellular growth and a 4-fold higher final product titer observed upon nutrient feeding. This first study of the relationship between the mTOR pathway and translational activity in CHO cultures provides key insights into the role of translational control in supporting greater productivity, which will lead to further enhancement of CHO cultures.

Control of ribosome traffic by position-dependent choice of synonymous codons

Messenger RNA (mRNA) encodes a sequence of amino acids by using codons. For most amino acids, there are multiple synonymous codons that can encode the amino acid. The translation speed can vary from one codon to another, thus there is room for changing the ribosome speed while keeping the amino acid sequence and hence the resulting protein. Recently, it has been noticed that the choice of the synonymous codon, via the resulting distribution of slow- and fast-translated codons, affects not only on the average speed of one ribosome translating the mRNA but also might have an effect on nearby ribosomes by affecting the appearance of ‘traffic jams’ where multiple ribosomes collide and form queues. To test this ‘context effect’ further, we here investigate the effect of the sequence of synonymous codons on the ribosome traffic by using a ribosome traffic model with codon-dependent rates, estimated from experiments. We compare the ribosome traffic on wild-type (WT) sequences and sequences where the synonymous codons were swapped randomly. By simulating translation of 87 genes, we demonstrate that the WT sequences, especially those with a high bias in codon usage, tend to have the ability to reduce ribosome collisions, hence optimizing the cellular investment in the translation apparatus. The magnitude of such reduction of the translation time might have a significant impact on the cellular growth rate and thereby have importance for the survival of the species.

Emerging therapeutic aspects in oncology

Cancer remains a peculiarly stubborn disease to treat. Some forms of cancer have seen tremendous advances in the effectiveness of their treatments, whereas other forms have remained resistant to pharmacological control. This lack of hope for success is in part due to the types of drugs that are used in the clinic, and the targeted biological system being based purely on cellular growth rates. However, recent drugs designed to affect specific signalling pathways or proteins have been showing much success. Thanks to the ingenuity of pharmacologists in understanding and targeting these processes, there have been real improvements in treatment. Here we are presented with some of the research into such critical systems that have to be understood, so that they can be conquered. We will also look at the challenges facing cancer pharmacologists and what the field may present to us all in the future. This article is part of a themed section on Emerging Therapeutic Aspects in Oncology. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-8.

Correlation of loss of spinophilin expression with increased proliferation and viability as a prognostic factor in patients with hepatocellular carcinoma.

e15064 Background: A loss of the putative tumorsuppressor protein spinophilin has been recently involved in the pathogenesis of lung and other carcinoma types. However, a detailed study of the clinical significance and functional cellular effects of spinophilin in hepatocellular carcinoma (HCC) has never been performed. Methods: In this study, we determined for the first time the expression pattern of spinophilin and some of it´s known interacting/down-stream genes (p53, p14ARF, pRB) and proliferation markers (MIB-1 and Cyclin D) in human hepatocellular carcinoma (HCC). The prognostic relevance of spinophilin expression was determined on a cohort of 104 HCC patients who underwent primary tumor resection, followed by functional gene silencing studies. Results: Reduced spinophilin expression was detected by three independent methods (Western blot, RT-PCR and IHC) in a substantial number of patients (70%, p&lt;0.05). Loss of spinophilin expression in HCC tissue was correlated with increased MIB-1 proliferation marker (p=0.01). In terms of clinical outcome, the disease-free survival (DFS) rate was 14.8% and 39.1% (p=0.010) and the cancer-specific survival (CSS) rate was 23.5% and 43.5% (p=0.006) for patients with low and high spinophilin expression, respectively. Multivariate Cox regression analysis identified low expression of spinophilin as an independent poor prognostic factor with respect to DFS (HR=1.8, CI=1.04-3.40, p=0.043) and CSS (HR=2.0, CI=1.1-3.8, p=0.025). Silencing of spinophilin expression in HepG2 cells was associated with increased cellular growth rate (p&lt;0.05). Conclusions: According to our data, the loss of this putative tumor suppressor protein is an independent negative prognosticator for the clinical outcome of HCC patients. Reduced levels of spinophilin expression are associated with higher proliferation rates of HCC cells.

In situ activity of a dominant Prochlorococcus ecotype (eHL‐II) from rRNA content and cell size

In the open ocean genetically diverse clades of the unicellular cyanobacteria Prochlorococcus are biogeographically structured along environmental gradients, yet little is known about their in situ activity. To address this gap, here we use the numerically dominant Prochlorococcus clade eHL-II (eMIT9312) as a model organism to develop and apply a method to examine their in situ activity using rRNA content and cell size as metrics of cellular physiology. For two representative isolates (MIT9312 and MIT9215) rRNA cell(-1) increases linearly with specific growth rate but is anticorrelated with cell size indicated by flow cytometrically measured (SSC). Although each strain has a unique relationship between cellular rRNA (or cell size) and growth rate, both strains have the same strong positive correlation between rRNA cell(-1) SSC(-1) and growth rate. We field test this approach and observe distinct patterns of eHL-II clade specific activity (rRNA cell(-1) SSC(-1)) with depth that are consistent with patterns of photosynthetic rates. This molecular technique provides unique insight into the ecology of Prochlorococcus and could potentially be expanded to include other microbes to unravel the ecological and biogeochemical contributions of genetically distinct marine side scatter microbes.

Low expression of the putative tumour suppressor spinophilin is associated with higher proliferative activity and poor prognosis in patients with hepatocellular carcinoma

Background:Spinophilin, a multifunctional intracellular scaffold protein, is reduced in certain types of cancer and is regarded as a novel putative tumour suppressor protein. However, the role of spinophilin in hepatocellular carcinoma (HCC) has never been explored before.Methods:In this study, we determined for the first time the expression pattern of spinophilin in human HCC by immunohistochemistry and quantitative reverse transcriptase–PCR analysis. In addition, we performed immunohistochemical analysis of p53, p14ARF and the proliferation marker Ki-67. Kaplan–Meier curves and multivariate Cox proportional models were used to study the impact on clinical outcome. Small interfering RNA (siRNA) was used to silence spinophilin and to explore the effects of reduced spinophilin expression on cellular growth.Results:In our study, complete loss of spinophilin immunoreactivity was found in 44 of 104 HCCs (42.3%) and reduced levels were found in an additional 37 (35.6%) cases. After adjusting for other prognostic factors, multivariate Cox regression analysis identified low expression of spinophilin as an independent prognostic factor with respect to disease-free (hazard ratio (HR)=1.8; 95% confidence interval (CI)=1.04–3.40; P=0.043) and cancer-specific survival (HR=2.0; CI=1.1–3.8; P=0.025). Reduced spinophilin expression significantly correlated with higher Ki-67 index in HCC (P=0.014). Reducing spinophilin levels by siRNA induced a higher cellular growth rate and increased cyclin D2 expression in tumour cells (P<0.05).Conclusion:This is the first study of the expression pattern and distribution of spinophilin in HCC. According to our data, the loss of spinophilin is associated with higher proliferation and might be useful as a prognostic marker in patients with HCC.

Abstract 3139: Identification and functional studies of p53 mutants detected in breast cancers after chemotherapy treatment.

Abstract Background: Approximately 4300 different TP53 mutations have been reported in human cancers. TP53 mutations, in particular those affecting the L2/L3 domains, are associated with resistance to anthracycline or mitomycin treatment in breast cancer patients. While many mutations have been characterised functionally, novel TP53 mutations are continuously reported. Here, we characterise 10 p53 protein variants encoded by mutated TP53 (5 within and 5 outside L2/L3) detected in locally advanced or metastatic breast cancers. Each tumour was previously characterised for response to therapy, allowing comparison between in vivo and in vitro findings. Methods: Mutated p53 variants were analysed for their ability to oligomerise with the wild-type protein and their subcellular localisation by immunoprecipitation and immunofluorescence, respectively. Their ability to induce transcription of target genes was determined by qPCR. Cellular growth rate, apoptosis and senescence were monitored by WST-1, TUNEL and beta-galactosidase assays, respectively. Results: Immunoprecipitation assays revealed each mutant protein to retain binding capacity for wild-type p53, thus potentially acting in a dominant negative manner. Even though each p53 variant located predominantly in the nucleus, the percentage of cells with only nuclear p53 localisation varied between 60-90%. None of the p53 variants were able to induce target genes to levels similar to wild-type p53, nor where they able to reduce cellular growth rate, induce apoptosis or senescence similar to wild-type p53 after anthracycline treatment in vitro. Conclusions/General Significance: Our data add further information characterising the effects of somatic TP53 mutations on p53 protein function and anthracycline resistance in breast cancer. Citation Format: Johanna Huun, Elisabet O. Berge, Johan R. Lillehaug, Per Eystein Lonning, Stian Knappskog. Identification and functional studies of p53 mutants detected in breast cancers after chemotherapy treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3139. doi:10.1158/1538-7445.AM2013-3139

Abstract 3775: Lysophosphatidic acid receptor-3 pathways are involved in up-regulation of cell migration and invasion activity of human sarcoma cells.

Abstract Lysophosphatidic acid (LPA) acts through G-protein coupled transmembrane receptors, and at least 6 types of LPA receptors (LPARs) have been identified. They regulate many different cellular responses, such as proliferation, survival, differentiation, migration, cytoskeletal changes and calcium influx through these pathways, and may act as a positive or negative regulator of cancer cell behaviors in a receptor specific manner which depends on cell types. Gene expression profiles analyzed in rat sarcoma model showed highly metastatic osteosarcoma cells expressing abundant LPA receptor-3 (LPAR3) compared to mesenchymal stem cells and non-metastatic MFH cells. In the current study, we generated LPAR3-knockdown cells from human fibrosarcoma HT1080 and osteosarcoma HOS cells by transfection with shRNA plasmids and investigated the involvement of LPA3 pathway for cell migration and invasion abilities. Both sarcoma cells expressed through LPAR1 to 5 except for LPAR1 in osteosarcoma HOS. Firstly, we tested the effects of chemical inhibitors of downstream effector of G-proteins, and results suggested that downstream of Gi and Gq protein inhibition suppressed cell motility in both sarcoma cells, while downstream of G12/13 inhibition did not. Then, we generated LPAR3 knockdown cells by transfection of shRNA for LPAR3, which resulted in effective knockdown of the LPAR3 expression in those cells. Expression of autotaxin, a mediator for LPA production, was suppressed in HT1080 but not in HOS by shRNA, indicating the difference of LPA production loop between those cells. LPAR3 knockdown did not affect cellular growth rates in both sarcoma cells. Moreover, LPA treatments stimulated cell proliferation in a dose dependent manner even in LPAR-3 knockdown cells, suggesting that LPA signaling on cell proliferation could be regulated by other LPAR pathways rather than LPAR3 in these cells. On the contrary, LPAR3-knockdown cells showed significantly lower cell motility than control cells both in cell motility assay and in scrape assay. Invasion assay with the Matrigel-coated filter also showed significantly lower invasion activities in LPAR3-knockdown HT1080 cells, which originally showed strong invasive capacity. While, there were no differences observed in matrix metalloproteinase (MMP) -2 and 9 activities on gelatin zymography, suggesting that the control of invasiveness through LPAR3 signaling pathways was independently regulated from MMPs. These results suggest that a part of the abilities for cell migration and invasion in human sarcoma cells were possibly regulated through the downstream of LPAR3, Gi and Gq protein signaling pathways, and those pathways could be the candidates for molecular targeted therapy against sarcomas. Further elucidation of detailed mechanisms underlying in these complex pathways will be required for future therapeutic approach. Citation Format: Kanya Honoki, Hiromasa Tsujiuchi, Akira Kido, Shinji Tsukamoto, Yasuhito Tanaka, Toshifumi Tsujiuchi. Lysophosphatidic acid receptor-3 pathways are involved in up-regulation of cell migration and invasion activity of human sarcoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3775. doi:10.1158/1538-7445.AM2013-3775

Abstract 3411: Mathematical model of the cell cycle to determine mechanism of action and optimize dosing schedule.

Abstract A mechanistic understanding of a drug's biological effect can have significant implications for selection of clinical dosing schedule, combination partners, as well as biomarker selection. In this context, a question often faced from target validation to the end of Phase I is how much of a drug's antitumor effect is through altering cell-cycle progression or through the induction of cell death. Here we demonstrate the utility of a mathematical model of the cell-cycle for determining the effect of a drug on cell-cycle kinetics, as an objective and quantiative interpretation of DNA flow cytometry and cellular growth rate data. The model uses periodic measurement of DNA content as analyzed by flow cytometry to determine the percentage of cells in G0/G1, S, and G2/M phases of the cell cycle, as well as continuous readouts of cell confluence over the course of treatment to assess growth rates. The model is then fitted to the data to extract effective cell cycle transition rates demonstrating the effect of the drug on cell cycle progression. To demonstrate of the approach, we applied the model to data acquired during treatment of A375 melanoma cells with the investigational drug TAK-733, an allosteric MEK 1/2 inhibitor hypothesized to cause a G1 arrest. Rather than an arrest at G1, the model predicted that the G1-to-S transition rate was reduced, and that much of the change in cell cycle data was due to an increase in the S-to-G2/M and G2/M-to-G0/G1 transition rates. These model predictions were then tested with video microscopy and both predictions (increased time spent in G0/G1 as well as reduced time spent in mitosis) were supported by experimental data. The model was then used to test whether the cell cycle activity of the compound has any implications for optimization of dosing schedule. By varying the schedule of drug treatment in the simulation, we find that for the biologically relevant concentration range the compound is schedule independent (i.e. effect is proportional to AUC). This finding suggests that there is no need to maintain constant pathway inhibition to see efficacy in tumor volume reduction. To test this prediction, we compare the results with the outcome of experiments in an A375 xenograft model with different dosing schedules and verify directly that AUC was indeed a better predictor of efficacy than Cmin. With this example, we demonstrate that mathematical modeling can be used to aid in interpretation of cell cycle data, often generated to describe the cell killing activity of oncology compounds. In addition, the model provides a method of interpreting the mechanism of action for early development decision making around schedule selection. This work demonstrates an approach that can be extended to other compounds to provide an integrated measure of the total contribution of cell death and/or cell cycle arrest, to help provide a mechanistic rationale for schedule selection with single agent and combination therapies. Citation Format: Jerome Mettetal, Derek Blair, Esha Gangoli, Patrick Vincent, Jeff Ecsedy, Wen Chyi Shyu, Arijit Chakravarty. Mathematical model of the cell cycle to determine mechanism of action and optimize dosing schedule. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3411. doi:10.1158/1538-7445.AM2013-3411

Minimal perfusion flow for osteogenic growth of mesenchymal stem cells on lattice scaffolds

A modeling approach to identify sets of culture conditions to promote homogeneous growth of cells in perfusion bioreactors equipped with regular shape scaffolds is proposed. We identify cases in which dynamic culturing is necessary using a zero‐dimensional mass transport and reaction model. Then, based on the three‐dimensional (3‐D) rendering of the flow field inside the bioreactor, we identify regions where cellular growth may become critical; finally, using a 1‐D mass transport and reaction model, we calculate the minimal perfusion flow necessary to maintain the cellular growth rate above a target threshold. The developed approach is used to analyze culturing conditions inside an indirect perfusion bioreactor equipped with a lattice scaffold. Regions where the perfusion flow is inadequate to foster cellular growth at the desired rate are identified. The perfusion flow required to maintain the target growth rate inside the bioreactor is calculated. © 2013 American Institute of Chemical Engineers AIChE J, 59: 3131–3144, 2013

Cell length sensing for neuronal growth control

Neurons exhibit great size differences, and must coordinate biosynthesis rates in cell bodies with the growth needs of different lengths of axons. Classically, axon growth has been viewed mainly as a consequence of extrinsic influences. However, recent publications have proposed at least two different intrinsic axon growth-control mechanisms. We suggest that these mechanisms form part of a continuum of axon growth-control mechanisms, wherein initial growth rates are pre-programmed by transcription factor levels, and subsequent elongating growth is dependent on feedback from intrinsic length-sensing enabled by bidirectional motor-dependent oscillating signals. This model might explain intrinsic limits on elongating neuronal growth and provides a mechanistic framework for determining the connections between genome expression and cellular growth rates in neurons.