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Roswell Park Memorial Institute medium, commonly referred to as RPMI, is a form of medium used in cell culture and tissue culture. It has traditionally been used for growth of Human lymphoid cells. This medium contains a great deal of phosphate and is formulated for use in a 5% carbon dioxide atmosphere. RPMI 1640 has traditionally been used for the serum-free expansion of human lymphoid cells.RPMI 1640 uses a bicarbonate buffering system and differs from most mammalian cell culture media in its typical pH 8 formulation.Properly supplemented with serum or an adequate serum replacement, RPMI 1640 allows the cultivation of many cell types, especially human T/B-lymphocytes, bone marrow cells and hybridoma cells.There are a variety of similar media in the RPMI series, such as RPMI 1640. Wikipedia.

Javaid S.,Harvard University | Zhang J.,Harvard University | Zhang J.,Roswell Park Memorial Institute | Zhang J.,Xian Jiaotong University | And 15 more authors.
Cell Reports

Epithelial-mesenchymal transition (EMT) is thought to contribute to cancer metastasis, but its underlying mechanisms are not well understood. To define early steps in this cellular transformation, we analyzed human mammary epithelial cells with tightly regulated expression of Snail-1, a master regulator of EMT. After Snail-1 induction, epithelial markers were repressed within 6hr, and mesenchymal genes were induced at 24hr. Snail-1 binding to its target promoters was transient (6-48hr) despite continued protein expression, and it was followed by both transient and long-lasting chromatin changes. Pharmacological inhibition of selected histone acetylation and demethylation pathways suppressed the induction as well as the maintenance of Snail-1-mediated EMT. Thus, EMT involves an epigenetic switch that may be prevented or reversed with the use of small-molecule inhibitors of chromatin modifiers. © 2013 The Authors. Source

Lilienfeld A.M.,Roswell Park Memorial Institute | Rogers M.,New York University
International Journal of Epidemiology

A continuum of reproductive causality is postulated, extending from fetal deaths - abortion, stillbirth, and neonatal - through a descending gradient of brain damage manifested in neuropsychiatric disorders. The research and administrative public health implications of these findings and the concept of the continuum are briefly but provocatively discussed. © The Author 2016; all rights reserved. Published by Oxford University Press on behalf of the International Epidemiological Association. Source

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Site: http://www.nature.com/nature/current_issue/

Unless mentioned otherwise, the experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. The fibroblast studies were performed on anonymized cells devoid of all identifiers. The data analysis involving urine orotic acid levels were performed under a protocol approved by the Institutional Review Board of Baylor College of Medicine. Urine samples were prepared by mixing 200 μl of with isotopic internal standard [15N ]orotic acid (Cambridge Isotope Laboratories). Orotic acid and orotidine were assayed on a Micromass Quattro mass spectrometer (Waters). HPLC was performed on a Waters ODS-AQ analytical column (150 mm × 2.0 mm internal diameter 5 μm bead size). Mobile phase was isocratic 0.05 M ammonium formate (pH 4.0). The MS–MS system was set at a flow rate of 0.2 ml min−1. The mass spectrometer was operated in electrospray ionization negative multiple-reaction monitoring mode. Nitrogen was used as nebulizer gas at a flow rate of 60–90 l h−1 and desolvation gas 500 l h−1. Other optimized mass spectrometer parameters were cone voltage 15 V, capillary 3,250 V and collision voltage 10 V. A metabolic network consisting of m metabolites and n reactions can be represented by a stoichiometric matrix S, where the entry S represents the stoichiometric coefficient of metabolite i in reaction j17. A constraint-based mode imposes mass balance, directionality and flux capacity constraints on the space of possible fluxes in the metabolic network’s reactions through a set of linear equations: where v stands for the flux vector for all of the reactions in the model (that is, the flux distribution). The exchange of metabolites with the environment is represented as a set of exchange (transport) reactions, enabling a pre-defined set of metabolites to be either taken up or secreted from the growth media. The steady-state assumption represented in equation (1) constrains the production rate of each metabolite to be equal to its consumption rate. Enzymatic directionality and flux capacity constraints define lower and upper bounds on the fluxes and are embedded in equation (2). In the following, flux vectors satisfying these conditions will be referred to as feasible steady-state flux distributions. The analyses were performed under the Roswell Park Memorial Institute Medium (RPMI)-1640m. We used the biomass function introduced in ref. 16. To determine the relation between ASS1 activity, CAD activity and growth rate, we used a generic human model and simulated the inactivation and activation of the reaction catalysed by ASS1. The inactivation was simulated by constraining the flux through the ASS1 reaction to zero, while the activation was simulated by enforcing increased positive flux through the ASS1 reaction up to the maximal possible flux, as computed via flux variability analysis17. At each such point, the maximal growth rate was computed via flux balance analysis17. Additionally, we estimated the flux through the reaction catalysed by CAD under maximal growth rate on the basis of 1,000 different feasible flux samples18. We next used genome-scale metabolic models for each of the NCI-60 cancer cell lines on the basis of their gene expression measurements10. In each cell-line model, we performed the following analyses. (1) We computed the production of each biomass component under both the inactivation and maximal activation of ASS1, as described above. The difference between the predicted production rates of each biomass component in the two states was then computed on the basis of the results of this optimization problem. (2) Similarly, we examined the flux change of each reaction under maximal biomass production in both the inactivation and activation states, as described above. In each of these states, we sampled the solution space and obtained 1,000 feasible flux distributions18. Focusing on the reactions associated with aspartate and glutamine, we computed the fold-change in flux rate together with its significance level. The latter was computed via a two-sided Wilcoxon rank-sum test. The largest fold-change among these reactions was predicted for the reactions catalysed by the CAD enzyme. For each tumour, normalized gene expression levels measured using RSEM19 were obtained from the RNASeqV2 data sets at the TCGA portal (https://tcga-data.nci.nih.gov/tcga/). Only matched tumour–normal pairs were used. For each tumour type, we computed the mean expression levels in the tumour and normal samples, added a pseudo-count of 1 to each mean and plotted the ratio between the means. Osteosarcoma or melanoma cell lines were seeded at 3 × 106 to 5 × 106 cells per 10 cm plate and incubated with either 4 mM l-glutamine, (α-15N, 98%, Cambridge Isotope Laboratories) for 24 h. Subsequently, cells were washed with ice-cold saline, lysed with 50% methanol in water and quickly scraped followed by three freeze–thaw cycles in liquid nitrogen. The insoluble material was pelleted in a cooled centrifuge (4 °C) and the supernatant was collected for consequent GC–MS analysis. Samples were dried under air flow at 42 °C using a Techne Dry-Block Heater with sample concentrator (Bibby Scientific) and the dried samples were treated with 40 μl of a methoxyamine hydrochloride solution (20 mg ml−1 in pyridine) at 37 °C for 90 min while shaking followed by incubation with 70 μl N,O-bis (trimethylsilyl) trifluoroacetamide (Sigma) at 37 °C for an additional 30 min. GC–MS. GC–MS analysis used a gas chromatograph (7820AN, Agilent Technologies) interfaced with a mass spectrometer (5975 Agilent Technologies). An HP-5ms capillary column 30 m × 250 μm × 0.25 μm (19091S-433, Agilent Technologies) was used. Helium carrier gas was maintained at a constant flow rate of 1.0 ml min−1. The GC column temperature was programmed from 70 to 150 °C via a ramp of 4 °C min−1, 250–215 °C via a ramp of 9 °C min−1, 215–300 °C via a ramp of 25 °C min−1 and maintained at 300 °C for an additional 5 min. The MS was by electron impact ionization and operated in full-scan mode from m/z = 30–500. The inlet and MS transfer line temperatures were maintained at 280 °C, and the ion source temperature was 250 °C. Sample injection (1 μl) was in splitless mode. Materials. Ammonium acetate (Fisher Scientific) and ammonium bicarbonate (Fluka) of LC–MS grade were used. Sodium salts of AMP, CMP, GMP, TMP and UMP were obtained from Sigma-Aldrich. Acetonitrile of LC grade was supplied from Merck. Water with resistivity 18.2 MΩ was obtained using Direct 3-Q UV system (Millipore). Extract preparation. The obtained samples were concentrated in speedvac to eliminate methanol, and then lyophilized tol dryness, re-suspended in 200 μl of water and purified on polymeric weak anion columns (Strata-XL-AW 100 μm (30 mg ml−1, Phenomenex)) as follows. Each column was conditioned by passing 1 ml of methanol, then 1 ml of formic acid/methanol/water (2/25/73) and equilibrated with 1 ml of water. The samples were loaded, and each column was washed with 1 ml of water and 1 ml of 50% methanol. The purified samples were eluted with 1 ml of ammonia/methanol/water (2/25/73) followed by 1 ml of ammonia/methanol/water (2/50/50) and then collected, concentrated in speedvac to remove methanol and lyophilized. Before LC–MC analysis, the obtained residues were re-dissolved in 100 μl of water and centrifuged for 5 min at 21,000 g to remove insoluble material. LC–MS analysis. The LC–MS/MS instrument consisted of an Acuity I-class UPLC system (Waters) and Xevo TQ-S triple quadruple mass spectrometer (Waters) equipped with an electrospray ion source and operated in positive ion mode was used for analysis of nucleoside monophosphates. MassLynx and TargetLynx software (version 4.1, Waters) were applied for the acquisition and analysis of data. Chromatographic separation was done on a 100 mm × 2.1 mm internal diameter, 1.8-μm UPLC HSS T3 column equipped with 50 mm × 2.1 mm internal diameter, 1.8-μm UPLC HSS T3 pre-column (both Waters Acuity) with mobile phases A (10 mM ammonium acetate and 5 mM ammonium hydrocarbonate buffer, pH 7.0 adjusted with 10% acetic acid) and B (acetonitrile) at a flow rate of 0.3 ml min−1 and column temperature 35 °C. A gradient was used as follows: for 0–6 min the column was held at 0% B, then 6–6.5 min a linear increase to 100% B, 6.5–7.0 min held at 100% B, 7.0–7.5 min back to 0% B and equilibration at 0% B for 2.5 min. Samples kept at 8 °C were automatically injected in a volume of 3 μl. For mass spectrometry, argon was used as the collision gas with a flow of 0.25 ml min−1. The capillary voltage was set to 2.90 kV, source temperature 150 °C, desolvation temperature 350 °C, desolvation gas flow 650 l min−1. Analytics were detected using multiple-reaction monitoring and applying the parameters listed in Supplementary Table 3. Single-molecule FISH (smFISH) was performed with probe libraries for Ass1 (74 probes, sequences described in Supplementary Methods) and Ki67 (96 probes20). Imaging was performed as previously described20. smFISH images were filtered with a Laplacian of Gaussian filter of size 15 pixels and standard deviation of 1.5 pixels. Each image is a maximum projection of ten stacks spaced 0.3 μm apart in the z-direction. Each dot in these figures represents a cell and the quantification dots were counted on eight z-stacks spaced 0.3 μm apart (total tissue volume 2.4 μm). Proximity ligation assay. The assay was performed as published21 using Sigma Aldrich kit (DUO 92004-30-RXN). Antibodies used for detection were diluted in PBS: ASS1 (1:200, ab170952, abcam), citrin (1:100, H00010165-M01, clone 4F4, abnova) and anti-CAD (1:100, ab40800, abcam). All cell lines were authenticated. Melanoma cell lines LOX IMVI and MALME-3m and osteosarcoma cell lines MNNG/HOS and U2OS were purchased from American Type Culture Collection (ATCC) and cultured using standard procedures in a 37 °C humidified incubator with 5% CO in RPMI (Invitrogen) supplemented with 10–20% heat-inactivated fetal bovine serum, 10% pen-strep and 2 mM glutamine. All cells are tested routinely for mycoplasma using a Mycoplasma EZ-PCR test kit (20–700-20, Biological Industries). MTT assay. Cells were seeded in 12-well plates at 4 × 104 to 8 × 104 cells per well in a triplicate. After 6 h for adherence of the cells, 0.1 mg ml-1 of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) (CAS 298-93-1, Calbiochem) in PBS was added to each cell type, starting at 0 h, in 24 h intervals. Deoxynucleotide Set (DNTP100-1KT, Sigma-Aldrich) was added to the cells’ medium first after adherence and then daily at a final concentration of 10 μM. Cells were lysed with dimethylsulfoxide (DMSO). Absorbance was measured at 570 nm. Crystal violet staining. Cells were seeded in 12-well plates at 40,000–100,000 cells per well in a triplicate. Time 0 was calculated as the time the cells became adherent, which was after about 6 h from plating. For each time point, cells were washed with PBS X1 and fixed in 4% PFA (in PBS). Cells were then stained with 0.1% Crystal Violet (C0775, Sigma-Aldrich) for 20 min (1 ml per well) and washed with water. Cells were then incubated with 10% acetic acid for 20 min with shaking. Extract was then diluted 1:4 in water and absorbance was measured at 590 nm every 24 h. Western blotting. Cells were lysed in RIPA (Sigma-Aldrich) and 0.5% protease inhibitor cocktail (Calbiochem). After centrifugation, the supernatant was collected and protein content was evaluated by the Bradford assay. One hundred micrograms from each sample under reducing conditions were loaded into each lane and separated by electrophoresis on a 10% SDS polyacrylamide gel. After electrophoresis, proteins were transferred to Immobilon transfer membranes (Tamar). Non-specific binding was blocked by incubation with TBST (10 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.1% Tween 20) containing 3% albumin from bovine serum for 1 h at 25 °C. Membranes were subsequently incubated with antibodies against ASS1 (1:500, sc-99178, Santa Cruz Biotechnology)22, p97 (1:10,000, PA5-22257, Thermo Scientific), GAPDH (1:1,000, 14C10, 2118, Cell Signaling)23, CAD (1:1,000, ab40800, abcam)24, phospho-CAD (Ser1859) (1:1,000, 12662, Cell Signaling)15, p70 S6 Kinase (1:1,000, 9202, Cell Signaling) and phospho-p70 S6 Kinase (Ser371) (1:1,000, 9208, Cell Signaling)25. Antibody was detected using peroxidase-conjugated AffiniPure goat anti-rabbit IgG or goat anti-mouse IgG (Jackson ImmunoResearch) and enhanced chemiluminescence western blotting detection reagents (EZ-Gel, Biological Industries). Gels were quantified by Gel Doc XR+ (BioRad) and analysed by ImageLab 4.1 software (BioRad). The band area was calculated by the intensity of the band. The obtained value was then divided by the value obtained from the loading control. RNA extraction and complementary DNA (cDNA) synthesis. RNA was extracted from cells by using PerfectPure RNA Cultured Cell Kit (5′-PRIME). cDNA was synthesized from 1 μg RNA by using qScript cDNA Synthesis Kit (Quanta). Quantitative PCR. Detection of ASS1 on cDNAs (see above) was performed using SYBR green PCR master mix (Tamar) and the required primers. Primer sequences were as follows. Human ASS1: forward, 5′-TTATAACCTGGGATGGGCACC-3′; reverse, 5′-TGGACATAGCGTCTGGGATTG-3′. Human HPRT: forward, 5′-ATTGACACTGGCAAAACAATGC-3′; reverse,: 5′-TCCAACACTTCGTGGGGTCC-3′. Analysis used StepOne real-time PCR technology (Applied Biosystems). Cells were seeded in 12-well plates at 30,000 cells per well, or in 10 cm plates at 106 cells per plate, in triplicate. The following day, cells were transfected with either 20 pmol or 600 pmol siRNA siGenome SMARTpool targeted to Citrin mRNA (M-007472-01, Thermo Scientific), respectively. Transfection was performed with Lipofectamine 2000 Reagent (11668-019, Invitrogen) in the presence of Opti-MEM I Reduced Serum Medium (31985-062, Invitrogen). Four hours after transfection, medium was replaced and experiments were performed starting 24 h after transfection. Over-expression. Cells were infected with pLenti3.3/TR and with pLenti6.3/TO/V5-DEST-based lentiviral vector with or without the human ASS1 transcript. Transduced cells were selected with 1 mg ml−1 Geneticin and with 7.5 μg ml−1 Blasticidin for each plasmid, respectively. When induction of expression was needed, cells were added with 10 μg ml−1 tetracycline/doxycycline. Cells were infected with pLKO-based lentiviral vector with or without the human ASS1 short hairpin RNA (shRNA) encoding one or two separate sequences combined (RHS4533-EG445, GE Healthcare, Dharmacon). Transduced cells were selected with 2 μg ml−1 puromycin. U2OS human osteosarcoma cell line was seeded in 6-well plates at 80,000 cells per well. The following day, cells were treated with either 100 nM rapamycin (R0395, Sigma-Aldrich) or with 10 μM 5FU (F6627, Sigma-Aldrich) in regular medium, with 10% dialysed FCS-arginine-free-RPMI (06-1104-34-1A, Biological Industries) or with both arginine-depleted medium and one of these drugs. Rapamycin and 5FU were renewed into the medium every day, whereas fresh arginine-free medium was supplemented twice a week. According to the approved IACUC protocol 17270415-2, tumours did not exceed the limits of more than 10% of the animal weight and were not longer than 1.5 cm in length in any dimension (Supplementary Fig. 2). Ten million MALME-3m melanoma cells suspended in 500 μl PBS with 5% Matrigel (4132053 Corning) were injected subcutaneously to 8- to 12-week-old male SCID mice that were purchased from Harlan. There were 22 SCID mice, from which 5 or 6 were used for each cell line in each of the three experiments performed. No randomization was used. Mice were monitored for survival and tumour burden twice a week by a veterinarian investigator who was blinded to the expected outcome. Tumours were measured using a calliper. After euthanization, tumours were removed and incubated in medium containing [15N]glutamine for 6 h followed by GC–MS analysis. Tumour size was calculated as published26. We used genome-scale metabolic models of NCI-60 cancer cell lines. The reconstruction method (on the basis of methods termed PRIME10 requires several key inputs: (1) the generic human model7; (2) gene expression data for each cell line19; and (3) growth rate measurements (available at the NCI website: https://dtp.cancer.gov/discovery_development/nci-60/cell_list.htm). The algorithm then reconstructs a specific metabolic model for each sample by modifying the upper bounds of reactions in accordance with the expression of the individual gene microarray values. Specifically, the model reconstruction process is as follows. (1) Decompose reversible reactions into unidirectional forward and backward reactions. (2) Evaluate the correlation between the expression of each reaction in the network and the measured growth rate. The expression of a reaction is defined as the mean over the expression of the enzymes catalysing it. (3) Modify upper bounds on reactions demonstrating significant correlation to the growth rate (after correcting for multiple hypothesis using false discovery rate) in a manner that is linearly related to expression value. All statistical analyses were performed using Tukey’s honest significant difference test or independent-samples Student’s t-test of multiple or two groups, respectively. Log-transformed data were used where differences in variance were significant and variances were correlated with means. The sample size was chosen in advance on the basis of common practice of the described experiment and is mentioned for each experiment. No statistical methods were used to predetermine sample size. Each experiment was conducted with biological and technical replicates and repeated at least three times unless specified otherwise. On the basis of pre-established criteria, individual outlier data points that were more than 2 standard deviations away from the mean were excluded from the data analysis. Statistical tests were done using Statsoft’s STATISTICA, version 10. All error bars are standard errors. P < 0.05 was considered significant in all analyses (*P < 0.05, **P < 0.005, ***P < 0.0005). Kaplan–Meier. For each cancer type, the Kaplan–Meier plot indicates the survival rates of the four different groups of patients as labelled. We analysed the cancer types for which there were sufficient survival data.

Cudkowicz G.,State University of New York at Buffalo | Cudkowicz G.,Roswell Park Memorial Institute | Bennett M.,State University of New York at Buffalo | Bennett M.,Roswell Park Memorial Institute
Journal of Immunology

Recipients of hemopoietic transplants are usually preexposed to whole body irradiation to deplete the blood-forming system, and so provide the transferred cells with a "graft bed" and stimuli for maximal proliferation and differentiation. It is generally assumed that irradiation in the lethal dose range would also be immunosuppressive and enable allogeneic cells to engraft. This assumption, however, is not correct. In the course of studies on the genetics of hybrid resistance to parental bone marrow grafts, it was noted that allografts did not establish themselves in irradiated hosts of certain mouse strains while succeeding in hosts of other strains (1). The mice in which marrow grafts grew and those in which the same cells failed sometimes belonged to inbred strains sharing the same H-2 alleles. This suggested that genes other than those specifying major transplantation antigens influenced the outcome of marrow allografts in irradiated mice. Additional evidence is now presented in support of this view. The data also indicate that a peculiar type of incompatibility for hemopoietic ailografts is common in irradiated mice, and that it results from destructive host anti-graft reactions. Since the processes of cellular proliferation, antibody formation, and skin graft rejection are impaired after whole body irradiation, bone marrow allografts are presumably rejected by a mechanism previously unknown. This type of allograft reaction is peculiar because it does not require proliferation of lymphoid cells and is tissue specific, thymus independent, and regulated by genetic factors which apparently do not affect the fate of other solid grafts. Source

Cudkowicz G.,The New School | Cudkowicz G.,Roswell Park Memorial Institute | Bennett M.,The New School | Bennett M.,Roswell Park Memorial Institute
Journal of Immunology

Mouse bone marrow cells transplanted across the major histocompatibility barrier fail to grow in given strain combinations even after exposure of prospective recipients to lethal doses of total body irradiation (1). The graft failures are presumably due to host reactivity against Hislocompalibility-2 (JI2)1 alloantigens which persists after irradiation and does not require presensitization or proliferation of host lymphoid cells. Other properties of this unusual allograft reactivity are its late maturation in infant mice at 3 wk of age, its apparent independence of thymic influence, and its regulation by immune response genes not linked to the H2 locus (1, 2). Cells of the bone marrow and other myeopoietic and lymphopoietic organs of inbred mice also fail to grow under conditions in which epithelial grafts are accepted, i.e., upon transplantation from parental-strain donors into irradiated F1 hybrid recipients (3-12). In most strain combinations, the resistance of F1 hybrids is attributable to heterozygosity at a locus closely linked to, or part of, the D end of 11-2, designated Hybrid-histocompatibility-1 or Hh-1 (8-10, 13, 14). The reactivity of the F1 mice is directed against cells bearing the products of hornozygous H/i-i alleles. Thus, the genetic determination of incompatibility f or bone marrow grafts could be different for parent-to-Fi and allogeneic cell transfers, but the immunobiology of graft rejection may still be the same in the two systems. The experiments described below address themselves to this question; the rcsults obtained indicate that hybrid resistance to marrow grafts of. Source

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