Cellular Growth Rate Research Articles

Maintenance of the hematopoietic stem cell pool in bone marrow niches by EVI1-regulated GPR56

Acute myeloid leukemia with high ecotropic viral integration site-1 expression (EVI1(high) AML) is classified as a refractory type of leukemia with a poor prognosis. To provide new insights into the prevention and treatment of this disease, we identified the high expression of EVI1-regulated G protein-coupled receptor 56 (GPR56), and the association of high cell adhesion and antiapoptotic activities in EVI1(high) AML cells. Knockdown of GPR56 expression decreased the cellular adhesion ability through inactivation of RhoA signaling, resulting in a reduction of cellular growth rates and enhanced apoptosis. Moreover, in Gpr56(-/-) mice, the number of hematopoietic stem cells (HSCs) was significantly decreased in the bone marrow (BM) and, conversely, was increased in the spleen, liver and peripheral blood. The number of Gpr56(-/-) HSC progenitors in the G0/G1-phase was significantly reduced and was associated with impaired cellular adhesion. Finally, the loss of GPR56 function resulted in a reduction of the in vivo repopulating ability of the HSCs. In conclusion, GPR56 may represent an important GPCR for the maintenance of HSCs by acting as a co-ordinator of interactions with the BM osteosteal niche; furthermore, this receptor has the potential to become a novel molecular target in EVI1(high) leukemia.

Developmental regulation of nucleolus size during Drosophila eye differentiation.

When cell cycle withdrawal accompanies terminal differentiation, biosynthesis and cellular growth are likely to change also. In this study, nucleolus size was monitored during cell fate specification in the Drosophila eye imaginal disc using fibrillarin antibody labeling. Nucleolus size is an indicator of ribosome biogenesis and can correlate with cellular growth rate. Nucleolar size was reduced significantly during cell fate specification and differentiation, predominantly as eye disc cells entered a cell cycle arrest that preceded cell fate specification. This reduction in nucleolus size required Dpp and Hh signaling. A transient enlargement of the nucleolus accompanied cell division in the Second Mitotic Wave. Nucleoli continued to diminish in postmitotic cells following fate specification. These results suggest that cellular growth is regulated early in the transition from proliferating progenitor cells to terminal cell fate specification, contemporary with regulation of the cell cycle, and requiring the same extracellular signals.

Targeting Aurora kinase A suppresses the growth of human oral squamous cell carcinoma cells in vitro and in vivo

Oncogene addiction has provided therapeutic opportunities in many human malignancies, but molecular targeted therapy for oral squamous cell carcinoma (OSCC) is not yet available. In this study, we attempted to identify an appropriate target molecule for treatment of patients with OSCC. Microarray analysis was performed to determine the gene expression profiles in nine human OSCC cell lines and a non-neoplastic keratinocyte cell line. The expression levels of Aurora kinase A (AURKA) mRNA and protein in human OSCC cells and tissues were examined. We investigated the effect of small interfering RNAs specific for AURKA (siAURKAs) and MLN8237, an AURKA selective inhibitor on the growth of OSCC cells in vitro and in vivo. We also analyzed clinical significance in AURKA mRNA expression levels in OSCC. AURKA was overexpressed in human OSCC cell lines and tissues. All siAURKAs almost completely suppressed the expression of AURKA protein, and significantly inhibited the growth of OSCC cells by 31-89%. MLN8237 also reduced the cellular growth rate by 38-74%. Both siAURKA and MLN8237 significantly reduced the size of subcutaneously xenografted OSCC tumors by 66% and 40%. Knockdown of AURKA expression and MLN8237 induced the growth inhibition of primary cultured cells established from patients' OSCC tumors. Furthermore, we found a significant association between AURKA mRNA expression levels and histological differentiation and lymph node metastasis. AURKA plays a critical role in the growth of human OSCC cells and targeting AURKA may be a useful therapeutic strategy for OSCC.

Small Changes in Enzyme Function can Lead to Surprisingly Large In Vivo Effects during Evolution

In principle, evolutionary outcomes could be largely predicted if all the relevant physicochemical variants of a particular protein function under selection were known and integrated into an appropriate physiological model. We have tested this principle by generating a family of variants of the tetracycline resistance protein TetX2 and identified the physicochemical properties most correlated with organismal fitness. Surprisingly, small changes in the Km(MCN), less than 2-fold, were sufficient to produce highly successful adaptive mutants over clinically relevant drug concentrations. We then built a quantitative model directly relating the in vitro physicochemical properties of the mutant enzymes to the growth rates of bacteria carrying a single chromosomal copy of the tet(X2) variants over a wide range of minocycline (MCN) concentrations. Importantly, this model allows the prediction of enzymatic properties directly from cellular growth rates as well as the physicochemical-fitness landscape of TetX2. Using experimental evolution and deep sequencing to monitor the allelic frequencies of the seven most biochemically efficient TetX2 mutants in 10 independently evolving populations, we showed that the model correctly predicted the success of the two most beneficial variants tet(X2)T280A and tet(X2)N371I. The structure of the most efficient variant, TetX2T280A, in complex with MCN at 2.7 A resolution suggests an indirect effect on enzyme kinetics. Taken together, these findings support an important role for readily accessible small steps in protein evolution that can, in turn, greatly increase the fitness of an organism during natural selection.

Functional characterisation of p53 mutants identified in breast cancers with suboptimal responses to anthracyclines or mitomycin

BackgroundApproximately 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. MethodsMutated 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. ResultsImmunoprecipitation 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% and 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. ConclusionsAll the 10 variants studied displayed inferior p53 functionality compared to the wild-type protein. General significanceOur data add further information characterising the effects of somatic TP53 mutations on p53 protein function and anthracycline resistance in breast cancer.

Small changes in enzyme function can lead to surprisingly large fitness effects during adaptive evolution of antibiotic resistance.

In principle, evolutionary outcomes could be largely predicted if all of the relevant physicochemical variants of a particular protein function under selection were known and integrated into an appropriate physiological model. We have tested this principle by generating a family of variants of the tetracycline resistance protein TetX2 and identified the physicochemical properties most correlated with organismal fitness. Surprisingly, small changes in the K(m(MCN)), less than twofold, were sufficient to produce highly successful adaptive mutants over clinically relevant drug concentrations. We then built a quantitative model directly relating the in vitro physicochemical properties of the mutant enzymes to the growth rates of bacteria carrying a single chromosomal copy of the tet(X2) variants over a wide range of minocycline (MCN) concentrations. Importantly, this model allows the prediction of enzymatic properties directly from cellular growth rates as well as the physicochemical-fitness landscape of TetX2. Using experimental evolution and deep sequencing to monitor the allelic frequencies of the seven most biochemically efficient TetX2 mutants in 10 independently evolving populations, we showed that the model correctly predicted the success of the two most beneficial variants tet(X2)(T280A) and tet(X2)(N371I). The structure of the most efficient variant, TetX2(T280A), in complex with MCN at 2.7 Å resolution suggests an indirect effect on enzyme kinetics. Taken together, these findings support an important role for readily accessible small steps in protein evolution that can, in turn, greatly increase the fitness of an organism during natural selection.

Rapid Antimicrobial Susceptibility Testing by Sensitive Detection of Precursor rRNA Using a Novel Electrochemical Biosensing Platform

Precursor rRNA (pre-rRNA) is an intermediate stage in the formation of mature rRNA and is a useful marker for cellular metabolism and growth rate. We developed an electrochemical sensor assay for Escherichia coli pre-rRNA involving hybridization of capture and detector probes with tail sections that are spliced away during rRNA maturation. A ternary self-assembled monolayer (SAM) prepared on gold electrode surfaces by coassembly of thiolated capture probes with hexanedithiol and posttreatment with 6-mercapto-1-hexanol minimized the background signal and maximized the signal-to-noise ratio. Inclusion of internal calibration controls allowed accurate estimation of the pre-rRNA copy number per cell. As expected, the ratio of pre-rRNA to mature rRNA was low during stationary phase and high during log phase. Pre-rRNA levels were highly dynamic, ranging from 2 copies per cell during stationary phase to ~1,200 copies per cell within 60 min of inoculation into fresh growth medium. Specificity of the assay for pre-rRNA was validated using rifampin and chloramphenicol, which are known inhibitors of pre-rRNA synthesis and processing, respectively. The DNA gyrase inhibitor, ciprofloxacin, was found to act similarly to rifampin; a decline in pre-rRNA was detectable within 15 min in ciprofloxacin-susceptible bacteria. Assays for pre-rRNA provide insight into cellular metabolism and are promising predictors of antibiotic susceptibility.

Direct in Vivo Evidence of Increased Chronic Lymphocytic Leukemia Cell Proliferation in Lymph Nodes Compared to Bone Marrow and Peripheral Blood

AbstractAbstract 184Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of mature B cells in peripheral blood (PB), bone marrow (BM) and lymph node (LN). Recent studies identified the LN as an important site of tumor cell activation and proliferation (Herishanu et al 2011). Using in vivo labeling of “newly-born” cells with deuterated water (2H2O; heavy water), the proliferation rate of CLL cells was estimated to range between 0.1 to >1% of the clone per day (Messmer et al 2005). Furthermore, a CXCR4dimCD5bright population of CLL cells in the PB contained more deuterium (2H)-labeled DNA and hence “recently-born cells than the CXCR4brightCD5dim population (Calissano et al 2011). Possible explanations for this observation include that CXCR4dimCD5bright cells proliferate more rapidly in the PB or that these cells are recent emigrants from tissues where proliferation occurs. As prior studies were performed on PB cells, the growth rates and characterization of the proliferative fraction of CLL cells in the LN remain unknown. Here we used 2H-labelling to directly compare cellular growth rates in PB, BM, and LN.Patients drank 2H2O for 28 days; on day 13 an excisional LN biopsy and a BM aspirate were obtained. PB samples were obtained at baseline and on days 13 and 28. CLL cells were isolated using positive selection, or sorted based on reciprocal differences in CXCR4 and CD5 density for isolation of “proliferative” (CXCR4dimCD5bright), “intermediate” (CXCR4intCD5int), and “resting” (CXCR4brightCD5dim) fractions of CLL clones. 2H incorporation into the DNA of newly divided cells was measured by mass spectrometry. Raw values were normalized to the 2H2O content in total body water. Cellular growth rates were calculated by dividing the fraction of 2H-labeled cells by the number of days from the start of the labeling period.To date, samples from 6 treatment-naïve CLL patients have been analyzed. On day 13, up to 16%, 9%, and 24% of the CLL cells sampled from PB, BM, and LN, respectively were 2H-labeled. The resulting mean estimated growth rate in % of the clone per day was for PB 0.41 (0.09 – 1.13), for BM 0.41 (0.28 – 0.68), and for LN 0.83 (0.31 – 1.84). The difference in growth rates between PB and LN was statistically significant (P<.02) and on average 2.5 times higher in the LN than in the PB. On day 28, the total fraction of 2H-labeled PB cells had further increased with the calculated growth rate in agreement with the growth rate in PB on day 13. CLL cells in the BM had a mean growth rate of 0.41% (0.28 – 0.68) of the clone per day, which was not significantly different than the growth rate in the PB. In fact in 2 patients the growth rate in the BM was lower than in the PB. The growth rates determined in LN and PB on day 13 were inversely correlated to the lymphocyte doubling time (r=-0.65 by Pearson correlation) and tended to be higher in patients with ZAP70 positive CLL. In keeping with the growth rates measured by 2H labeling, the fraction of Ki67-expressing CLL cells was higher in the LN than in the PB [% cells mean ± SD (PB = 3.8 ± 1.6, LN = 18.4 ± 3.1; P=.005)]. Interestingly, while the average CLL growth rate in LNs by 2H-labeling was 2.5-times the rate in PB, the Ki-67 positive fraction in LNs was 5-times the fraction in the PB, further supporting the view that active clonal proliferation occurs predominantly in the LN, from which recently-born cells enter the PB. Consistent with a prior study of PB CLL cells (Calissano et al 2011), the CXCR4dimCD5bright subset in LN exhibited higher 2H-labeling than the bulk of the clonal cells (intermediate fraction) and the resting fraction. These studies indicate that a CXCR4dimCD5bright subset exists in LN-resident CLL cells and has higher 2H-labeling than the rest of the clone. Specifically, the calculated growth rate of the CXCR4dimCD5bright subset was on average 3.2-times the growth rate of all CLL cells in the LN. Moreover, the data suggest that sampling the PB for newly-born cells is a reliable measure of the degree of proliferation occurring in LNs.Taken together our data show that the proliferation rate of CLL cells is higher in the LN than in the BM and PB and suggest that some of the newborn cells exit the LN within days. A clonal subset of CXCR4dimCD5bright cells is present in both the LN and PB and might harbor the proliferative core of the disease. Disclosures:Gatmaitan:KineMed: Employment. Emson:KineMed: Employment. Chiorazzi:KineMed: Dr. Chiorazzi holds stock options in KineMed, Inc. Other.

Competition between conjugation and M13 phage infection in Escherichia coli in the absence of selection pressure: a kinetic study.

Inter- and intraspecies horizontal gene transfer enabled by bacterial secretion systems is a powerful mechanism for bacterial genome plasticity. The type IV secretion system of Escherichia coli, encoded by the F plasmid, enables cell-to-cell contact and subsequent DNA transfer known as conjugation. Conjugation is compromised by phage infection that specifically targets the secretion machinery. Hence, the use of phages to regulate the spread of genes, such as acquired antibiotic resistance or as general biosanitation agents, has gained interest. To predict the potential efficacy, the competition kinetics must first be understood. Using quantitative PCR to enumerate genomic loci in a resource-limited batch culture, we quantify the infection kinetics of the nonlytic phage M13 and its impact on conjugation in the absence of selection pressure (isogenic set). Modeling the resulting experimental data reveals the cellular growth rate to be reduced to 60% upon phage infection. We also find a maximum phage infection rate of 3×10−11 mL phage−1 min−1 which is only 1 order of magnitude slower than the maximum conjugation rate (3×10−10 mL cell−1 min−1), suggesting phages must be in significant abundance to be effective antagonists to horizontal gene transfer. In the regime where the number of susceptible cells (F+) and phages are equal upon initial infection, we observe the spread of the conjugative plasmid throughout the cell population despite phage infection, but only at 10% of the uninfected rate. This has interesting evolutionary implications, as even in the absence of selection pressure, cells maintain the ability to conjugate despite phage vulnerability and the associated growth consequences.

Analysis of cell viability using time-dependent increase in fluorescence intensity

We report the use of resazurin (AlamarBlue) dye in a robust assay for cell viability of primary cells. Human mononuclear cells were used here for immunological studies, but the method can be applied to monitor reduction potential of any living cell. Reduction of AlamarBlue dye is widely used in several commercial assays of cell viability. Although it is fast and easy with immortal cell lines, the method is impractical for the primary cells due to their slower metabolic activity. We propose that the viability of human primary cells can be determined with AlamarBlue by monitoring the increase in fluorescence intensity in a matter of a few hours. In the presence of AlamarBlue, the dynamic increase in cellular reduction capacity is linear for several hours or, alternatively, the assay can be repeated to monitor the viability at any time point of cell culture. In addition to testing cellular growth rates and cytotoxicity, the application can be used to compare sample quality of cells that have been frozen or represent a pool of multiple donors. This application of the AlamarBlue cell viability assay is simple, rapid, and cost-effective, and therefore it is also well suited for high-throughput studies.

Non-colony type monolayer culture of human embryonic stem cells

Regenerative medicine, relying on human embryonic stem cell (hESC) technology, opens promising new avenues for therapy of many severe diseases. However, this approach is restricted by limited production of the desired cells due to the refractory properties of hESC growth in vitro. It is further hindered by insufficient control of cellular stress, growth rates, and heterogeneous cellular states under current culture conditions. In this study, we report a novel cell culture method based on a non-colony type monolayer (NCM) growth. Human ESCs under NCM remain pluripotent as determined by teratoma assays and sustain the potential to differentiate into three germ layers. This NCM culture has been shown to homogenize cellular states, precisely control growth rates, significantly increase cell production, and enhance hESC recovery from cryopreservation without compromising chromosomal integrity. This culture system is simple, robust, scalable, and suitable for high-throughput screening and drug discovery.

NITROGEN LIMITATION INCREASES BREVETOXINS IN KARENIA BREVIS (DINOPHYCEAE): IMPLICATIONS FOR BLOOM TOXICITY(1).

Laboratory and field measurements of the toxin content in Karenia brevis cells vary by >4-fold. These differences have been largely attributed to genotypic variations in toxin production among strains. We hypothesized that nutrient limitation of growth rate is equally or more important in controlling the toxicity of K. brevis, as has been documented for other toxic algae. To test this hypothesis, we measured cellular growth rate, chlorophyll a, cellular carbon and nitrogen, cell volume, and brevetoxins in four strains of K. brevis grown in nutrient-replete and nitrogen (N)-limited semi-continuous cultures. N-limitation resulted in reductions of chlorophyll a, growth rate, volume per cell and nirtogen:carbon (N:C) ratios as well as a two-fold increase (1%-4% to 5%-9%) in the percentage of cellular carbon present as brevetoxins. The increase in cellular brevetoxin concentrations was consistent among genetically distinct strains. Normalizing brevetoxins to cellular volume instead of per cell eliminated much of the commonly reported toxin variability among strains. These results suggest that genetically linked differences in cellular volume may affect the toxin content of K. brevis cells as much or more than innate genotypic differences in cellular toxin content per unit of biomass. Our data suggest at least some of the >4-fold difference in toxicity per cell reported from field studies can be explained by limitation by nitrogen or other nutrients and by differences in cell size. The observed increase in brevetoxins in nitrogen limited cells is consistent with the carbon:nutrient balance hypothesis for increases in toxins and other plant defenses under nutrient limitation.

Protective Effects of Extracts and Flavonoids Isolated from Scutia buxifolia Reissek against Chromosome Damage in Human Lymphocytes Exposed to Hydrogen Peroxide

Flavonoids are claimed to protect against cardiovascular disease, certain forms of cancer and ageing, possibly by preventing initial DNA damage. Therefore, we investigated the protective effects of crude extract, ethyl acetate fraction and flavonoids (quercetin, quercitrin, isoquercitrin and rutin) isolated from the leaves from Scutia buxifolia against chromosome damage induced by H2O2 in human lymphocytes by analyzing cellular growth rate, cell viability, mitotic index and chromosomal instability. We found a differential response among the compounds tested, with the ethyl acetate fraction being more effective than the crude extract, a difference perhaps related to the presence of the antioxidants identified and quantified by HPLC/DAD. In general, quercetin, isoquercitrin and rutin recovered the mitotic index and chromosomal instability more than quercitrin after treatment with hydrogen peroxide.

An in silico platform for the design of heterologous pathways in nonnative metabolite production

BackgroundMicroorganisms are used as cell factories to produce valuable compounds in pharmaceuticals, biofuels, and other industrial processes. Incorporating heterologous metabolic pathways into well-characterized hosts is a major strategy for obtaining these target metabolites and improving productivity. However, selecting appropriate heterologous metabolic pathways for a host microorganism remains difficult owing to the complexity of metabolic networks. Hence, metabolic network design could benefit greatly from the availability of an in silico platform for heterologous pathway searching.ResultsWe developed an algorithm for finding feasible heterologous pathways by which nonnative target metabolites are produced by host microorganisms, using Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae as templates. Using this algorithm, we screened heterologous pathways for the production of all possible nonnative target metabolites contained within databases. We then assessed the feasibility of the target productions using flux balance analysis, by which we could identify target metabolites associated with maximum cellular growth rate.ConclusionsThis in silico platform, designed for targeted searching of heterologous metabolic reactions, provides essential information for cell factory improvement.

Abstract 1643: MicroRNA processing genes and implications for complex traits

Abstract Given the fundamental role of microRNA (miRNA) expression in physiological, developmental and pathological processes, we hypothesize that genes involved in miRNA biogenesis and processing are also important in human complex traits. We identified fifty two such genes and evaluated their expression for their roles in cellular growth and sensitivity to various chemotherapeutic agents in a set of 120 International HapMap cell lines. Gene expression data were obtained through microarray-based gene expression profiling (Affymetrix GeneChip® Human Exon 1.0 ST array) and high-throughput sequencing of the transcriptome (RNA-Seq); data on sensitivity to chemotherapeutic drugs carboplatin, cisplatin, daunorubicin and etoposide were queried using a publicly available pharmacogenomics resource we developed (www.PACdb.org). We found 14 (27%) and 24 (47%) of these miRNA processing gene expression correlated with cellular growth rate and at least one of the 4 chemotherapeutic sensitivities, respectively. Regression analysis for expression between miRNA processing genes and genome-wide miRNAs (quantified using Exiqon miRCURYTM LNA arrays) suggests that the miRNA processing genes affect miRNAs and downstream target genes, leading to complex traits variability. Furthermore, from the results of genome-wide association studies we conducted between genetic variations (in the form of single nucleotide polymorphisms (SNPs) and copy number variations (CNVs)) and miRNA processing genes, we demonstrated that genetic variations that affect the expression of these miRNA processing genes as potential expression quantitative trait loci (eQTLs) also have downstream effects on human complex phenotypes, including cellular growth, sensitivity to chemotherapy and disease susceptibility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1643. doi:1538-7445.AM2012-1643

Intensification of high cell-density cultivations of rE. coli for production of S. pneumoniae antigenic surface protein, PspA3, using model-based adaptive control

This work proposes an innovative methodology to control high density fed-batch cultures of E. coli, based on measurements of the concentration of dissolved oxygen and on estimations of the cellular specific growth rate (µ), of the yield of biomass/limiting substrate (Y (xs)) and of the maintenance coefficient (m). The underlying idea is to allow cells to grow according to their metabolic capacity, without the constraints inherent to pre-set growth rates. Cellular concentration was assessed on-line through a capacitance probe. Three configurations of the control system were compared: (1) pre-set value for the three control parameters; (2) continuously updating µ; (3) updating µ, Y (xs) and m. Implementation of an efficient noise filter for the signal of the capacitance probe was essential for a good performance of the control system. The third control strategy, within the framework of an adaptive model-based control, led to the best results, with biomass productivity reaching 9.2g(DCW)/L/h.

The Variable Subdomain of Escherichia coli SecA Functions To Regulate SecA ATPase Activity and ADP Release

Bacterial SecA proteins can be categorized by the presence or absence of a variable subdomain (VAR) located within nucleotide-binding domain II of the SecA DEAD motor. Here we show that VAR is dispensable for SecA function, since the VAR deletion mutant secAΔ519-547 displayed a wild-type rate of cellular growth and protein export. Loss or gain of VAR is extremely rare in the history of bacterial evolution, indicating that it appears to contribute to secA function within the relevant species in their natural environments. VAR removal also results in additional secA phenotypes: azide resistance (Azi(r)) and suppression of signal sequence defects (PrlD). The SecAΔ(519-547) protein was found to be modestly hyperactive for SecA ATPase activities and displayed an accelerated rate of ADP release, consistent with the biochemical basis of azide resistance. Based on our findings, we discuss models whereby VAR allosterically regulates SecA DEAD motor function at SecYEG.

Mixed Effects Modeling of Proliferation Rates in Cell-Based Models: Consequence for Pharmacogenomics and Cancer

The International HapMap project has made publicly available extensive genotypic data on a number of lymphoblastoid cell lines (LCLs). Building on this resource, many research groups have generated a large amount of phenotypic data on these cell lines to facilitate genetic studies of disease risk or drug response. However, one problem that may reduce the usefulness of these resources is the biological noise inherent to cellular phenotypes. We developed a novel method, termed Mixed Effects Model Averaging (MEM), which pools data from multiple sources and generates an intrinsic cellular growth rate phenotype. This intrinsic growth rate was estimated for each of over 500 HapMap cell lines. We then examined the association of this intrinsic growth rate with gene expression levels and found that almost 30% (2,967 out of 10,748) of the genes tested were significant with FDR less than 10%. We probed further to demonstrate evidence of a genetic effect on intrinsic growth rate by determining a significant enrichment in growth-associated genes among genes targeted by top growth-associated SNPs (as eQTLs). The estimated intrinsic growth rate as well as the strength of the association with genetic variants and gene expression traits are made publicly available through a cell-based pharmacogenomics database, PACdb. This resource should enable researchers to explore the mediating effects of proliferation rate on other phenotypes.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract A2: miR-10a regulation of PIK3CA and response to taxol in non small cell lung cancer

Abstract Phosphatidylinositol 3-kinases (PI3Ks) are a family of enzymes involved in diverse cellular functions including cell growth, proliferation, differentiation, motility, survival and apoptosis. PI3K allows for the transmission of extracellular cues to an intracellular response via its activation through receptor tyrosine kinase or k-ras. Activation of this pathway is antagonized by the phosphatase PTEN. This signaling pathway is under extremely tight regulation and even slight perturbations can lead to aberrant pathway activation, as is the case in a majority of cancers. We are interested in whether PIK3CA is regulated by one or more microRNAs and whether those miRNAs have a therapeutic effect on cancer cell viability and drug response in PI3K-driven oncogenesis. In a systematic approach to identifying miRNA inhibitors with significant effects on cell viability and response to paclitaxel in NSCLC, we performed a high-throughput screen and identified several candidate microRNAs. Regulatory targets of candidate miRNAs were identified through a combination of in vitro and in silico approaches. Targets were then validated using qRT-PCR, protein quantification, and luciferase reporter assays. The response of cancer cells to perturbations of the candidate miRNA was assessed through flow cytometric analysis of cell cycle phase distribution, colony formation and caspase activation assays. The inhibitor screen revealed that inhibition of miR-10a increased cellular growth rate and resistance to paclitaxel. Further validation demonstrated a 10-fold increase in cell viability in the presence of miR-10a inhibitor, and a 10-fold decrease in the presence of miR-10a mimic. Manipulation of miR-10a levels in either direction resulted in significant changes in both mRNA and protein levels of its predicted target, the catalytic subunit of phosphatidylinositol 3-kinase (PI3K), which was confirmed to be a direct target by luciferase reporter assay. Increasing miR-10a levels inhibited the ability to form colonies, while decreasing miR-10a levels increased colony formation. To assess the prognostic value of miR-10a we assessed expression in NSCLC patients and observed that high miR-10a levels correlate with longer overall survival. The identification of a miR-10a as a modulator of cellular response to taxanes, and PI3K as a regulatory target which mediates that response, define a novel regulatory pathway modulating paclitaxel sensitivity in lung cancer, which may provide novel adjuvant strategies along with paclitaxel in the treatment of lung cancer and may also provide biomarkers for predicting paclitaxel response in NSCLC. Ultimately this study will provide new insights into the molecular basis of lung cancer and a new set of potential therapeutic targets for this devastating disease. Citation Format: Chris DeSevo, Liqin Du, Carmen Behrens, Ignacio I. Wistuba, John Minna, Alexander Pertsemlidis. miR-10a regulation of PIK3CA and response to taxol in non small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A2.