Cells In Exponential Growth Research Articles

Domoic Acid Oxidative Effects on the Microalgae Phaeodactylum tricornutum.

Domoic acid (DA) is a natural occurring marine biotoxin. Oxidative stress generation due to DA exposure was reported in animals, but little is known on the phytoplankton community. The aim of this work was to verify whether exposure to DA in the marine diatom Phaeodactylum tricornutum favors reactive oxygen species (ROS) generation in the intracellular environment modifying its antioxidant capacity. Active species production, non-enzymatic antioxidant content, and antioxidant enzyme activities over the three growth phases of P. tricornutum exposed to 64 µM DA were evaluated. Results obtained in exponential growing cells showed a time-depending seven-fold increase in the 2',7' dichlorofluorescein diacetate dye oxidation rate. Superoxide dismutase and catalase activities showed a two-fold increase, and glutathione related enzymes activities were also significantly increased in treated diatoms as compared to controls. However, glutathione and ascorbate contents significantly decreased after incubation of the cells with DA. Similar effects were observed in latent and stationary phases of cell development. These results showed that DA could cause a severe oxidant-dependent impact on a non-toxic algae.

Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p-coumarate

Pseudomonas putida KT2440 is a well-studied bacterium for the conversion of lignin-derived aromatic compounds to bioproducts. The development of advanced genetic tools in P. putida has reduced the turnaround time for hypothesis testing and enabled the construction of strains capable of producing various products of interest. Here, we evaluate an inducible CRISPR-interference (CRISPRi) toolset on fluorescent, essential, and metabolic targets. Nuclease-deficient Cas9 (dCas9) expressed with the arabinose (8K)-inducible promoter was shown to be tightly regulated across various media conditions and when targeting essential genes. In addition to bulk growth data, single cell time lapse microscopy was conducted, which revealed intrinsic heterogeneity in knockdown rate within an isoclonal population. The dynamics of knockdown were studied across genomic targets in exponentially-growing cells, revealing a universal 1.75 ± 0.38 h quiescent phase after induction where 1.5 ± 0.35 doublings occur before a phenotypic response is observed. To demonstrate application of this CRISPRi toolset, β-ketoadipate, a monomer for performance-advantaged nylon, was produced at a 4.39 ± 0.5 g/L and yield of 0.76 ± 0.10 mol/mol from p-coumarate, a hydroxycinnamic acid that can be derived from grasses. These cultivation metrics were achieved by using the higher strength IPTG (1K)-inducible promoter to knockdown the pcaIJ operon in the βKA pathway during early exponential phase. This allowed the majority of the carbon to be shunted into the desired product while eliminating the need for a supplemental carbon and energy source to support growth and maintenance.

Step-by-Step Regulation of Productive and Abortive Transcription Initiation by Pyrophosphorolysis

An understanding of the kinetics and mechanism of bacterial transcription initiation is needed to understand regulation of gene expression and advance fields from antibiotic discovery to promoter design. The step-by-step forward kinetics and mechanism of initiation and RNA-DNA hybrid growth, made irreversible by omitting pyrophosphate (PPi) byproduct, were determined recently for E. coli RNA polymerase (RNAP)-λPR promoter complexes. Strong position-dependences of overall rate constants (kcat/Km analogs) for each nucleotide-addition step were observed because of coupling of hybrid growth to disruption of promoter contacts, bubble closing, and RNAP escape. Here we investigate reversal of these steps (pyrophosphorolysis) at PPi concentrations ([PPi]) found in exponentially-growing cells. We quantify [PPi] effects on the amount and rate of synthesis of long (>10-mer, post-escape) and short (stalled, abortive) RNA to determine how PPi regulates initiation. Physiological [PPi] makes uridine incorporation and some other initiation steps significantly reversible. Physiological [PPi] reduces the fraction of RNAP-promoter complexes that productively initiate and the rate of RNA synthesis per productive complex, while increasing the fraction of complexes that abortively initiate, affecting abortive rates, and shifting the abortive-product distribution to shorter RNAs. Pyrophosphorolysis rates for some initiation complexes are orders of magnitude larger than for removal of the same nucleotide from elongation complexes because of the strong bias toward the pre-translocated state in initiation, and exhibit even stronger dependences on nucleotide identity (pyrimidine ≫ purine). Because cytoplasmic [PPi] is much higher in exponential-phase than stationary-phase cells, these [PPi] effects on initiation rates and amounts of RNA synthesis must be physiologically-relevant.

Long Non-Coding Transcript in LEF1 Locus Is a Novel Regulator of Cell Cycle and Apoptotic Pathways in Myeloid Leukemic Cells

Myelodysplastic syndrome and acute myeloid leukemia are hematologic disorders characterized by inefficient differentiation and aberrant control of cell growth and apoptosis. In early MDS, blasts, unable to differentiate, exhibit exacerbated proliferation and apoptosis. However, MDS progression and AML transformation are associated with a progressive resistance to apoptosis. The molecular mechanisms that control these cellular decisions are not fully understood. Long non-coding RNAs (lncRNA) have gained great attention in recent years as they have been implicated in a wide variety of biological functions. Here we focus on a long non-coding transcript encoded in the LEF1 locus as a novel regulator of apoptosis and proliferation signaling in hematologic disease.Our previous work using microarray technology identified a long non-coding RNA, antisense to LEF1 gene, which expression was reduced in myelodysplastic syndrome patients. We mapped the sequence to a previously characterized transcript, LEF1-AS1. This transcript is highly conserved and has been recently associated to B-cell differentiation. We observed a modulation of LEF1-AS1 during the first stages of in-vitro erythroid differentiation from CD34+ HSC to erythroblast (from collection day 6 to day 8 after addition of erythropoietin, n=6, 78% mean reduction, P<0.0001).Due to reported reduction on LEF1-AS1 expression in MDS and low endogenous expression of LEF1-AS1 in myeloid cells lines, we decided to overexpress the transcript in myeloid cell lines HL60 and K562 to study the function of the lncRNA. Stably transfected cells were obtained by DMRIE-C mediated transfection using pcDNA vector containing full length lef1-AS1 or empty pcDNA vector. Selection was carried out by addition of geneticin to the culture medium.Unexpectedly, after stable over-expression of LEF1-AS1, we could not detect any alteration of LEF1 expression in the mRNA or protein level in both myeloid cell lines (HL60 and K562). Despite of the lack of regulatory effect upon LEF1, we observed a significant inhibition in cell proliferation evaluated Carboxyfluorescein succinimidyl ester (CFSE) labelling (p<0.05).To investigate the antiproliferative effects of LEF1-AS1, we analyzed the cell cycle phase distribution of the stably transfected cell HL60 by flow cytometry using propidium iodide DNA staining. LEF1-AS1 over-expression resulted in a significant increase in the proportion of S-phase cells (p=0.01) and no difference in G2/M, suggesting intra S-phase imprisonment.Additionally, we evaluated the effect of LEF1-AS1 over-expression upon apoptosis in stably transfected HL60 using annexin/Propidium Iodide Apoptosis flow cytometry assay. We observed no difference in the apoptosis level between LEF1-AS1-overexpressing cells and control cells in basal culture conditions; however, LEF1-AS1 over-expression inhibited significantly serum deprivation-induced apoptosis (reduction of 50%, p<0.05).We screened several apoptosis and proliferation regulators by RT-PCR and western blotting and detected a strong increase in anti-apoptotic BCL2 and CDKN1A/p21 in HL60 cells over-expressing LEF1-AS1. This increase was observed in cells in exponential growth and starvation. Interestingly, we did not observe this antiapoptotic effect in K562, a cell line that do not express BCL2 endogenously.To confirm that the phenotypical effects of lef1-AS over -expression were not mediated by LEF1 function, we also over-expressed lef1-AS1 in Hela, a cell line lacking endogenous expression of LEF1. Empty vector and lef1-AS1-Hela stably-transfected-cells had no detectable expression of LEF1; however, a similar inhibition of cell proliferation and S-phase accumulation were observed.To further investigate the mechanism behind these phenotypical effects we carried out a RNA-pulldown experiment coupled with mass spectrometry to identify the proteins that bind to LEF1-AS1. Our preliminary results identified an apoptosis regulator protein in association to this transcript and we are currently working to unravel the implications of this interaction.Remarkably, BCL2 is simultaneously antiapoptotic and anti-proliferative and S-phase arrest has been associated to p21 up-regulation. Our results depict LEF1-AS1 as a fine-tuner of proliferation and survival decisions, acting, at least partially, through modulation of BCL2 and CDKN1A/p21. DisclosuresNo relevant conflicts of interest to declare.

Adaptive responses of Kluyveromyces marxianus CCT 7735 to 2-phenylethanol stress: Alterations in membrane fatty-acid composition, ergosterol content, exopolysaccharide production and reduction in reactive oxygen species

2-phenylethanol (2-PE) is a higher aromatic alcohol with a rose-like aroma used in the cosmetic and food industries as a flavoring and displays potential for application as an antifungal. Biotechnological production of 2-PE from yeast is an interesting alternative due to the non-use of toxic compounds and the generation of few by-products. Kluyveromyces marxianus CCT 7735 is a thermotolerant strain capable of producing high 2-PE titers from L-Phenylalanine; however, like other yeast species, its growth has been strongly inhibited by this alcohol. Herein, we aimed to evaluate the effect of 2-PE on cell growth, cell viability, membrane permeability, glucose uptake, metabolism, and morphology in K. marxianus CCT 7735, as well as its adaptive responses. The stress condition was imposed after 4 h of cultivation by adding 3.0 g.L−1 of 2-PE in exponential growing cells. 2-PE stress impaired yeast growth, glucose uptake, fermentative metabolism, membrane permeability, and cell viability. Moreover, the stress condition provoked changes in both morphology and surface roughness. The reactive oxygen species (ROS) increased immediately on exposure to 2-PE. Changes in membrane fatty-acid composition, ergosterol content, exopolysaccharides production, and reduction of the ROS levels appear to be the result of adaptive responses in K. marxianus. Our results provided insights into a better understanding of the effects of 2-PE on K. marxianus and its adaptive responses.

Radiation-induced cell cycle perturbations: a computational tool validated with flow-cytometry data

Cell cycle progression can be studied with computational models that allow to describe and predict its perturbation by agents as ionizing radiation or drugs. Such models can then be integrated in tools for pre-clinical/clinical use, e.g. to optimize kinetically-based administration protocols of radiation therapy and chemotherapy. We present a deterministic compartmental model, specifically reproducing how cells that survive radiation exposure are distributed in the cell cycle as a function of dose and time after exposure. Model compartments represent the four cell-cycle phases, as a function of DNA content and time. A system of differential equations, whose parameters represent transition rates, division rate and DNA synthesis rate, describes the temporal evolution. Initial model inputs are data from unexposed cells in exponential growth. Perturbation is implemented as an alteration of model parameters that allows to best reproduce cell-cycle profiles post-irradiation. The model is validated with dedicated in vitro measurements on human lung fibroblasts (IMR90). Cells were irradiated with 2 and 5 Gy with a Varian 6 MV Clinac at IRCCS Maugeri. Flow cytometry analysis was performed at the RadBioPhys Laboratory (University of Pavia), obtaining cell percentages in each of the four phases in all studied conditions up to 72 h post-irradiation. Cells show early {text{G}}_{2}-phase block (increasing in duration as dose increases) and later {text{G}}_{1}-phase accumulation. For each condition, we identified the best sets of model parameters that lead to a good agreement between model and experimental data, varying transition rates from {text{G}}_{1}- to S- and from {text{G}}_{2}- to M-phase. This work offers a proof-of-concept validation of the new computational tool, opening to its future development and, in perspective, to its integration in a wider framework for clinical use.

In Memoriam: Marvin A. Van Dilla: 1919-2019.

© 2020 International Society for Advancement of Cytometry.

Efficient computation of stochastic cell-size transient dynamics

BackgroundHow small, fast-growing bacteria ensure tight cell-size distributions remains elusive. High-throughput measurement techniques have propelled efforts to build modeling tools that help to shed light on the relationships between cell size, growth and cycle progression. Most proposed models describe cell division as a discrete map between size at birth and size at division with stochastic fluctuations assumed. However, such models underestimate the role of cell size transient dynamics by excluding them.ResultsWe propose an efficient approach for estimation of cell size transient dynamics. Our technique approximates the transient size distribution and statistical moment dynamics of exponential growing cells following an adder strategy with arbitrary precision.ConclusionsWe approximate, up to arbitrary precision, the distribution of division times and size across time for the adder strategy in rod-shaped bacteria cells. Our approach is able to compute statistical moments like mean size and its variance from such distributions efficiently, showing close match with numerical simulations. Additionally, we observed that these distributions have periodic properties. Our approach further might shed light on the mechanisms behind gene product homeostasis.

The effects of copper on photosynthesis and biomolecules yield in Chlorolobion braunii.

Our knowledge of the effects of copper on microalgal physiology is largely based on studies conducted with high copper concentrations; much less is known when environmentally relevant copper levels come into question. Here, we evaluated the physiology of Chlorolobion braunii exposed to free copper ion concentrations between 5.7×10-9 and 5.0×10-6 mol · L-1 , thus including environmentally relevant values. Population growth and maximum photosynthetic quantum yield of PSII were determined daily during the 96h laboratory controlled experiment. Exponentially-growing cells (48h) were analyzed for effective quantum yield and rapid light curves (RLC), and total lipids, proteins, carbohydrates, chlorophyll a and carotenoids were determined. The results showed that growth rates and population density decreased gradually as copper increased in experiment, but the photosynthetic parameters (maximum and effective quantum yields) and photochemical quenching (qP) decreased only at the highest free copper concentration tested (5.0×10-6 mol · L-1 ); nonphotochemical quenching (NPQ) increased gradually with copper increase. The RLC parameters Ek and rETRmax were inversely proportional to copper concentration, while α and Im decreased only at 5.0×10-6 mol · L-1 . The effects of copper in biomolecules yield (mg · L-1 ) varied depending on the biomolecule. Lipid yield increased at free copper concentration as low as 2.5×10-8 mol · L-1 , but proteins and carbohydrates were constant throughout.

Resistance of early stationary phase E. coli to membrane permeabilization by the antimicrobial peptide Cecropin A

Antimicrobial peptides (AMPs) cause bacterial membrane permeabilization and ultimately cell death at low μM concentrations. The membrane permeabilization action of a moth derived AMP Cecropin A on E. coli cells in exponential growth (mid-log phase) is well studied. At 1× MIC concentration, Cecropin A penetrates the lipopolysaccharide (LPS) barrier and causes outer membrane (OM) and cytoplasmic membrane (CM) permeabilization. For non-septating cells, permeabilization of both membranes begins at one pole. For septating cells, OM permeabilization begins at the septal region and CM permeabilization begins at one pole. However, in nature bacteria are frequently found in nutrient-starved conditions. Here we extend our single-cell microscopy assays to the attack of Cecropin A on E. coli cells in early stationary phase. Stationary phase E. coli is much more resistant to membrane permeabilization by Cecropin A than mid-log phase E. coli. A tenfold higher concentration of Cecropin A is required to observe CM permeabilization in the majority of stationary phase cells, and even then permeabilization proceeds more slowly. In addition, the spatial pattern of initial CM permeabilization changes from localized at one pole to global. Studies of lipid mutant strains suggest that a sufficient localized concentration of the anionic phospholipid phosphatidylglycerol (PG) guides the position of initial attack of the cationic AMP Cecropin A on the CM.

Cellular Responses of Chlorococcum Sp. Algae Exposed to Zinc Oxide Nanoparticles by Using Flow Cytometry

Evaluation of 50 nm zinc oxide nanoparticles’ (ZnO-NPs) effects on the microalgae Chlorococcum sp. growing in high salt growth medium (HSM) was investigated by using flow cytometry parameters (cell size (FSC), granularity (SSC), chlorophyll a fluorescence (FL3), and formation of reactive oxygen species (ROS)). Algal cells in exponential growth were exposed to 0–100 mg/L of ZnO-NPs and their physiological responses were measured after 24 and 96 h of treatment. Behavior of ZnO-NPs was analyzed in HSM and results indicated that ZnO-NPs formed agglomeration with a large distribution. Total soluble Zn concentration increased when initial ZnO-NP concentration increased. Significant negative effect on algal cells was observed after 96 h exposition and at high ZnO-NP concentration. This negative impact was evaluated by the significant increase in ROS production, inhibition in the photosynthetic electron transport, and reduction in cell growth. In this study, using flow cytometry multi-parameters might help to prevent and evaluate inhibitory effect of oxide nanoparticles on aquatic photosynthetic microorganisms.

Toward the Quantitative Characterization of Arginine Phosphorylations in Staphylococcus aureus.

The Gram-positive bacterium Staphylococcus aureus plays an important role as an opportunistic pathogen and causative agent of nosocomial infections. As pathophysiological research gained insights into host-specific adaptation and a broad range of virulence mechanisms, S. aureus evolved as a model organism for human pathogens. Hence the investigation of staphylococcal proteome expression and regulation supports the understanding of the pathogenicity and relevant physiology of this organism. This study focused on the analysis of protein regulation by reversible protein phosphorylation, in particular, on arginine residues. Therefore, both proteome and phosphoproteome of S. aureus COL wild type were compared with the arginine phosphatase deletion mutant S. aureus COL ΔptpB under control and stress conditions in a quantitative manner. A gel-free approach, adapted to the special challenges of arginine phosphorylations, was applied to analyze the phosphoproteome of exponential growing cells after oxidative stress caused by sublethal concentrations of H2O2. Together with phenotypic characterization of S. aureus COL ΔptpB, this study disclosed first insights into the physiological role of arginine phosphorylations in Gram-positive pathogens. A spectral library based quantification of phosphopeptides finally allowed us to link arginine phosphorylation to staphylococcal oxidative stress response, amino acid metabolism, and virulence.

PO-128 MicroRNA-449A enhances the radiosensitivity of prostate cancer cells

IntroductionMicroRNAs (miRNAs) are evolutionarily conserved, endogenous, small noncoding RNAs that regulate the stability and translation of target mRNA by primarily binding to the 3′ -UTR18. MiRNAs have been reported to be involved in DNA damage response induced by ionising radiation (IR). c-Myc is reduced when cells treated with IR or other DNA damaging agents. However, it is unknown whether miRNAs participate in c-Myc downregulation in response to IR. MicroRNA-449a is deregulated in various types of cancers, including prostate cancer.Material and methodsThe human prostate cancer lines, LNCaP, PC-3 and DU-145 cells in exponential growth were irradiated by x-rays at room temperature. Overexpressing miR-499a and miR-con was generated by plasmid transduction using GV214. MiR-449a antagomir (anti-miR-449a) and non-targeting sequence were synthesised. Transfection was performed with Trans IT −2020 Transfection Reagent. The cell viability assay was carried out basis on thiazolyl blue tetrazolium blue (MTT). Cell proliferation was analysed using Cell Counting Kit-8(CCK-8). Cell cycle analysis was acquired using FACS Calibur flow cytometer. MiRNAs and mRNA expression were determined using quantitative RT-PCR analysis. Protein expression were analysed by western blotting.Results and discussionsIn the present study, we found that miR-449a was upregulated and c-Myc was downregulated in response to IR in LNCaP cells. Overexpression of miR-449a or knockdown of c-Myc promoted the sensitivity of LNCaP cells to IR. By establishing c-Myc as a direct target of miR-449a, we revealed that miR-449a enhanced radiosensitivity by repressing c-Myc expression in LNCaP cells. Moreover, we showed that miR-449a enhanced radiation-induced G2/M phase arrest by directly downregulating c-Myc, which controlled the Cdc2/CyclinB1 cell cycle signal by modulating Cdc25A. In summary, our study reported here demonstrated that miR-449a enhanced the sensitivity of LNCaP cells to IR by directly targeting c-Myc, which controlled the Cdc2/cyclinB1 cell cycle signal by modulating Cdc25A/Rb/E2F pathway. Furthermore, we found that both miR-449a and c-Myc responded to irradiation and either overexpression of miR-449a or knockdown of c-Myc sensitised LNCaP cells to irradiation.ConclusionThese findings highlighted an unrecognised mechanism of miR-449a-mediated c-Myc regulation in response to IR, which provides a support for the combination of ionising radiation with miRNAs regulation as a therapeutic strategy for patients with prostate cancer.

Cell-free protein synthesis from non-growing, stressed Escherichia coli

Cell-free protein synthesis is a versatile protein production system. Performance of the protein synthesis depends on highly active cytoplasmic extracts. Extracts from E. coli are believed to work best; they are routinely obtained from exponential growing cells, aiming to capture the most active translation system. Here, we report an active cell-free protein synthesis system derived from cells harvested at non-growth, stressed conditions. We found a downshift of ribosomes and proteins. However, a characterization revealed that the stoichiometry of ribosomes and key translation factors was conserved, pointing to a fully intact translation system. This was emphasized by synthesis rates, which were comparable to those of systems obtained from fast-growing cells. Our approach is less laborious than traditional extract preparation methods and multiplies the yield of extract per cultivation. This simplified growth protocol has the potential to attract new entrants to cell-free protein synthesis and to broaden the pool of applications. In this respect, a translation system originating from heat stressed, non-growing E. coli enabled an extension of endogenous transcription units. This was demonstrated by the sigma factor depending activation of parallel transcription. Our cell-free expression platform adds to the existing versatility of cell-free translation systems and presents a tool for cell-free biology.

Abstract PR08: Combinatorial CRISPR-Cas9 reveals many cancer synthetic lethal interactions are private to cell type

Abstract We developed a systematic approach to map human genetic networks by combinatorial CRISPR-Cas9 perturbation coupled to robust analysis of growth kinetics. We targeted all pairs of 73 cancer genes with multiplexed guide RNAs in two cell lines, testing 23,652 combinations. Numerous therapeutically relevant interactions were identified, most private to one cell line. These patterns replicated with combinatorial drugs at 80% precision. Thus, cellular context will be critical to synthetic-lethal therapies. Here, we combined multiplex targeting with array-based oligonucleotide synthesis to create dual-gRNA libraries covering up to 105 defined gene pairs. We conducted genetic interaction screens by transducing the dual-gRNA lentiviral library into a population of cells stably expressing Cas9, maintaining these cells in exponential growth over the course of four weeks, then sampling the relative changes in gRNAs at days 3, 14, 21 and 28 post-transduction. To robustly quantify gene fitness and genetic interactions, we developed a computational analysis framework that integrates all samples across the multiple days of the experiment. Using this method we evaluated all pairwise gene knockout combinations among a panel of 73 genes divided between tumor-suppressor genes (TSG) and cancer-relevant drug targets (DT), a subset of which were also verified oncogenes. Experiments were performed in two cancer cell lines: HeLa, a cervical cancer cell line driven by Human Papilloma Virus (HPV); and A549, a lung cancer cell line driven by KRAS G12S mutation. With nine gRNA pairs per combination, the library comprised 23,652 double gene knockout constructs and 657 single gene constructs; testing two replicates in each cell line yielded a total of 94,608 unique tests of interaction. Measurements of gene fitness (fg) were well correlated between biological replicates in the same cell line; Pearson r=0.96, p = 4.2×(10)^(-40), as were the π scores for significant genetic interactions; r = 0.75 p = 7.7×(10)^(-12). Moreover, we observed a significant correlation between the number of genetic interactions identified for a gene and its single gene fitness (HeLa: r = 0.77, p = 3.4×(10)^(-10); A549: r = 0.45, p = 0.0018), suggesting that network hubs may have increased functional importance to cancer cells relative to genes with fewer interactions; such a relationship has been previously observed in model organisms but not before in humans5. Interestingly, we found that the genetic interactions identified from these data were remarkably different between cell lines. A total of 97 synthetic-lethal (negative) genetic interactions were identified in either HeLa or A549 cells. Of these, only 12 were identified in both cell lines, while the remaining 85 interactions were private to a cell line (HeLa: 40 of 52, A549: 45 of 57). We next sought to validate these findings, and in particular the discrepancies across cell lines. We selected ten pairs of DT genes for which a synthetic-lethal genetic interaction had been identified in only one of the two cell lines. Rather than simply reproduce the dual CRISPR knockout experiment (gene-gene interaction), our goal was to examine the viability of cells exposed to drugs inhibiting the corresponding gene products (drug-drug interaction). In total, drug-drug assays validated eight of ten interactions when tested in the cell line for which the interaction had been first observed by dual CRISPR (80% precision or positive predictive value). In contrast, when the same ten gene pairs were tested in the other cell line that had not been implicated by dual CRISPR knockout, only two showed a negative genetic interaction by drug-drug assay (80% negative predictive value). Thus, the differences in genetic interaction across cell lines seen by systematic CRISPR could be largely reproduced as drug-drug interactions in small-scale assays. In summary, we have introduced a combinatorial CRISPR-Cas9 genetic interaction mapping technology that successfully identifies many therapeutically-relevant genetic interactions in cancer and shows the great importance of cellular context on the architecture of the genetic interaction network. Recognizing that there will be great diversity in genetic interaction between different tumors it will be important to perform these studies across a large number of samples, which is enabled by the high-throughput method we have developed. Citation Format: John Paul Shen, Dongxin Zhao, Roman Sasik, Jens Luebeck, Amanda Birmingham, Ana Bojorquez-Gomez, Jason Kreisberg, Trey Ideker, Prashant Mali. Combinatorial CRISPR-Cas9 reveals many cancer synthetic lethal interactions are private to cell type [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr PR08.

Abstract 5430: GAPDH loss in a tumorigenic human glioblastoma cell line

Abstract Background: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a ubiquitous, multifunctional 37 kDa enzyme best known for catalyzing the sixth step in glycolysis. GAPDH expression is so consistent that it has been granted “housekeeping” status, and is used as a standard (along with β-actin) for normalizing Western blots. While increased GAPDH expression has been described in hypoxia, diabetes, and some cancers, decreased expression is rare. Here we describe a new human glioblastoma cell line that exhibits GAPDH loss, while maintaining a high degree of malignancy. Methods: Primary cultures of human glioblastoma cells were initiated and maintained in DMEM with 10% FBS. This particular cell line (designated E297) grew readily, and was passaged more than 40 times. E297 cells were confirmed to be negative for HIV, HTLV, hepatitis B and C, CMV, and mycoplasma contamination. Cells in exponential growth were stained for routine markers, and studied by DNA flow cytometry. GAPDH expression was studied by Western blot analysis using polyclonal rabbit anti-human GAPDH IgG (Abcam, Rosemont, IL), with HeLa, U138, U87 and U251 cells (as well as β-actin staining) as positive controls. For intracranial implantation into Wistar-Furth rats (male, 10-11 weeks), cells were suspended at 20 x 106 cells/ml. Using aseptic technique, rats were anesthetized, and placed into a stereotactic frame. A burr hole was drilled 3 mm right of the bregma and 25 µl of suspension injected (5 mm depth) over 10 min. by Hamilton syringe. Rats were studied by MRI and euthanized when symptomatic. Results: E297 cells have glial/epithelioid morphology, with a doubling time of 24 + 2 hours in logarithmic growth phase, with a single DNA subpopulation with 28% S phase. They stain positively for GFAP, vimentin, bFGF, c-myc and p53, but fail to express GAPDH. 10/10 animals implanted intracerebrally developed tumors, becoming symptomatic and requiring sacrifice at 25 days (mean). Loss of GAPDH expression was confirmed by Western blot analysis. Conclusions: The E297 human glioblastoma cell line is highly aggressive and proliferates rapidly. It is tumorigenic in nonimmunosuppressed rats but lacks GAPDH protein. We conclude that GAPDH expression is not essential for glioblastoma cell proliferation under routine culture conditions, and care needs to be taken when using GAPDH as a normalization standard for Western blots of cancer cells. Citation Format: Zachary Gaertner, Yi Lu, Jinsuh Kim, Gayatry Mohapatra, Ankit Mehta, Sanjani Lakka, Herbert Engelhard. GAPDH loss in a tumorigenic human glioblastoma cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5430. doi:10.1158/1538-7445.AM2017-5430

Abstract 3039: Combinatorial CRISPR-Cas9 gene knockout to enable genetic interaction mapping in human cells

Abstract Genetic interactions, in particular negative or ‘synthetic-lethal’ interactions for which simultaneous disruption of two genes causes cell killing, have implications for therapeutic development. The feasibility of this approach has been demonstrated with the recent approval of the drug olaparib, an inhibitor of PARP1, specifically for tumors with loss-of-function mutations in BRCA1/2. However, beyond olaparib, further applications of synthetic-lethal cancer therapy have been limited by poor understanding of the important genetic interactions in a cancer cell, and how these vary from one cancer type to another or from patient to patient. To enable systematic mapping of these genetic interaction networks, we developed a CRISPR-Cas9 screening methodology for knocking out single and pairs of genes in high-throughput. Here, we combined multiplex targeting with array-based oligonucleotide synthesis to create dual-gRNA libraries covering up to 10^5 defined gene pairs. In these libraries, each construct bears two gRNAs, with each gRNA designed to target either a gene or a scrambled non-targeting sequence absent from the genome. We conducted genetic interaction screens by transducing the dual-gRNA lentiviral library into a population of cells stably expressing Cas9, maintaining these cells in exponential growth over the course of four weeks, then sampling the relative changes in gRNAs at days 3, 14, 21 and 28 post-transduction. To robustly quantify gene fitness and genetic interactions, we developed a computational analysis framework that integrates all samples across the multiple days of the experiment. Using this method we evaluated all pairwise gene knockout combinations among a panel of 73 genes divided between tumor-suppressor genes (TSG) and cancer-relevant drug targets (DT); including negative controls this amounted to a total of 23,652 combinations. Experiments were performed in three cancer cell lines: HeLa, A549 and 293T. In total 162 therapeutically relevant interactions were identified, of which 146 (90.1%) were private to one cell line. None of the interactions were observed in all three cell lines. These patterns replicated in low throughput assays with combinatorial drugs at 80% precision. In summary, we have introduced a combinatorial CRISPR-Cas9 genetic interaction mapping technology that successfully identifies many therapeutically-relevant genetic interactions in cancer and shows the great importance of cellular context on the architecture of the genetic interaction network. Recognizing that there will be great diversity in genetic interaction between different tumors it will be important to perform future studies across a large number of samples, which is enabled by the high-throughput method we have developed. Citation Format: John Paul Shen, Dongxin Zhao, Roman Sasik, Jens Luebeck, Ana Bojorquez-Gomez, Katherine Licon, Trey Ideker, Prashant Mali. Combinatorial CRISPR-Cas9 gene knockout to enable genetic interaction mapping in human cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3039. doi:10.1158/1538-7445.AM2017-3039

Functional analyses of putative PalS (Palindromic Self-recognition) motifs in bacterial Hfq

The bacterial protein Hfq has been linked to nucleic acid metabolism and signaling, however its explicit role has been elusive. Recently it was proposed that the C-termini of Hfq subunits in Hfq6 complexes could be involved in functional interactions with other Hfq hexamers and/or nucleic acids. To test the proposed model of the native Hfq complex experimentally, we genetically engineered chimeric Hfq6 complexes, in which C-termini of bacterial Hfq subunits were substituted with a sequence derived from human histone H2B (hH2B) that includes multiple functionally significant amino acids whose modifications have been linked to carcinogenesis. We demonstrate that this substitution results in an enhanced formation of dodecameric assemblies by the Hfq–hH2B hybrid – a result pointing to the possibility of a (functional) homology between these motifs in proteins from distant kingdoms. We hypothesize that these putative Palindromic Self-recognition (PalS) motifs could act as proteins' ‘cohesive ends’ that could allow the protein complexes carrying such motifs to interact dynamically and dissociate–reassociate in response to stress and/or growth phase-specific changes. We provide experimental support to the latter hypothesis and demonstrate that in E. coli the dodecameric Hfq assemblies are formed in a growth stage-specific manner. We describe a refined system – consisting solely of purified Hfq, polynucleotide phosporylase (PNP) and ADP – that allows reconstitution in vitro of characteristic ‘SDS-insensitive’ Hfq6–Hfq6 assemblies observed in experiments with whole-cell extracts obtained from exponentially-growing cells. We also optimized conditions for the extraction of intact native dodecameric Hfq complexes.

Response of irradiated and bystander cells to charged particles in two-dimensional and three-dimensional colon models

The radiation-induced bystander effect, wherein unirradiated cells near to or sharing medium with irradiated cells express biological responses, most often has been studied in two-dimensional monolayer cultures, although some studies with three-dimensional models and in vivo have also shown bystander signaling. We have shown previously that DNA damage, measured as foci of the DNA repair-related protein 53BP1, occurs in unirradiated bystander cells in a three-dimensional skin epithelium model irradiated with protons or iron ions (Lumpkins et al., submitted). In the current work, we extend the studies to a second epithelial model, colon, with studies in both two-dimensional monolayer and a three-dimensional tissue model using Caco2 human colon epithelial cancer cells and AG01522 human fibroblasts.For the monolayer studies, Caco2 cells in exponential growth were irradiated then co-cultured, sharing medium in an insert system, with unirradiated cells. Cells were irradiated with 250 kVp X-rays at Massachusetts General Hospital or with 1 GeV/amu protons, silicon ions or iron ions at the National Space Radiation Laboratory at Brookhaven National Laboratory. At varying times after irradiation, cell damage was assayed as micronuclei (MN) induction or formation of 53BP1 foci in both irradiated and bystander cells. For the three-dimensional studies, AG01522 fibroblasts were embedded in a collagen gel, then Caco2 cells were grown on the top of the gel. Each three-dimensional construct was cut in half prior to irradiation, with one half irradiated then immediately placed in contact with the other, bystander, half for co-culture. At selected times after irradiation, irradiated and bystander construct halves were fixed and sectioned, and 53BP1 foci were counted.In monolayer culture, irradiated Caco2 cells showed a dose-dependent increased fraction of cells with MN after exposure to X-rays, protons, iron ions or silicon ions. Bystander Caco2 cells sharing medium with the irradiated cells also showed an increased fraction of cells with MN, reaching similar levels of ∼16% cells with MN, about a threefold increase over controls, after 1 Gy of all types of radiation. The fraction of cells with 53BP1 foci depended on radiation type and time of assay after irradiation, with the induction of foci generally greatest 5 h after irradiation and increasing with radiation dose. In bystander Caco2 cells, the appearance of foci generally was delayed, with the maximal fraction of cells showing foci at 12 h. In three-dimensional culture, after X-ray or proton exposure, cells showed similar trends to those seen in two-dimensional growth, i.e. with both the Caco2 and the AG01522 cells, the fraction of irradiated cells having 53BP1 foci reached a maximum at 5 h, but with bystander cells, the maximum occurred at 12 h after irradiation. This delay in the appearance of foci in bystander cells compared with irradiated cells is similar to our findings in the three-dimensional skin model composed of keratinocytes and fibroblasts.In summary, our data now show in two different epithelial tissue models in both two-dimensional and three-dimensional models, radiation-stimulated intercellular signaling results in substantial levels of DNA damage in unirradiated cells. Because Caco2 cells are a carcinoma cell line, the studies are now being extended to a three-dimensional colon model using normal human colonic epithelial cells.

Population Dynamics of Metastable Growth-Rate Phenotypes

Neo-Darwinian evolution has presented a paradigm for population dynamics built on random mutations and selection with a clear separation of time-scales between single-cell mutation rates and the rate of reproduction. Laboratory experiments on evolving populations until now have concentrated on the fixation of beneficial mutations. Following the Darwinian paradigm, these experiments probed populations at low temporal resolution dictated by the rate of rare mutations, ignoring the intermediate evolving phenotypes. Selection however, works on phenotypes rather than genotypes. Research in recent years has uncovered the complexity of genotype-to-phenotype transformation and a wealth of intracellular processes including epigenetic inheritance, which operate on a wide range of time-scales. Here, by studying the adaptation dynamics of genetically rewired yeast cells, we show a novel type of population dynamics in which the intracellular processes intervene in shaping the population structure. Under constant environmental conditions, we measure a wide distribution of growth rates that coexist in the population for very long durations (>100 generations). Remarkably, the fastest growing cells do not take over the population on the time-scale dictated by the width of the growth-rate distributions and simple selection. Additionally, we measure significant fluctuations in the population distribution of various phenotypes: the fraction of exponentially-growing cells, the distributions of single-cell growth-rates and protein content. The observed fluctuations relax on time-scales of many generations and thus do not reflect noisy processes. Rather, our data show that the phenotypic state of the cells, including the growth-rate, for large populations in a constant environment is metastable and varies on time-scales that reflect the importance of long-term intracellular processes in shaping the population structure. This lack of time-scale separation between the intracellular and population processes calls for a new framework for population dynamics which is likely to be significant in a wide range of biological contexts, from evolution to cancer.