Roswell Park Memorial Institute
Roswell Park Memorial Institute
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.
Lilienfeld A.M.,Roswell Park Memorial Institute |
Rogers M.,New York University
International Journal of Epidemiology | Year: 2016
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.
News Article | February 22, 2017
Pre-B acute lymphoblastic leukaemia (ALL) cells were obtained from patients who gave informed consent in compliance with the guidelines of the Internal Review Board of the University of California San Francisco (Supplementary Table 2). Leukaemia cells from bone marrow biopsy of patients with ALL were xenografted into sublethally irradiated NOD/SCID (non-obese diabetic/severe combined immunodeficient) mice via tail vein injection. After passaging, leukaemia cells were collected. Cells were cultured on OP9 stroma cells in minimum essential medium-α (MEMα; Invitrogen), supplemented with 20% fetal bovine serum (FBS), 2 mM l-glutamine, 1 mM sodium pyruvate, 100 IU/ml penicillin and 100 μg/ml streptomycin. Primary chronic myeloid leukaemia (CML) cases were obtained with informed consent from the University Hospital Jena in compliance with institutional internal review boards (including the IRB of the University of California San Francisco; Supplementary Table 3). Cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Invitrogen) supplemented with 20% BIT serum substitute (StemCell Technologies); 100 IU/ml penicillin and 100 μg/ml streptomycin; 25 μmol/l β-mercaptoethanol; 100 ng/ml SCF; 100 ng/ml G-CSF; 20 ng/ml FLT3; 20 ng/ml IL-3; and 20 ng/ml IL-6. Human cell lines (Supplementary Table 2) were obtained from DSMZ and were cultured in Roswell Park Memorial Institute medium (RPMI-1640; Invitrogen) supplemented with GlutaMAX containing 20% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin. Cell cultures were kept at 37 °C in a humidified incubator in a 5% CO atmosphere. None of the cell lines used was found in the database of commonly misidentified cell lines maintained by ICLAC and NCBI Biosample. All cell lines were authenticated by STR profiles and tested negative for mycoplasma. BML275 (water-soluble) and imatinib were obtained from Santa Cruz Biotechnology and LC Laboratories, respectively. Stock solutions were prepared in DMSO or sterile water at 10 mmol/l and stored at −20 °C. Prednisolone and dexamethasone (water-soluble) were purchased from Sigma-Aldrich and were resuspended in ethanol or sterile water, respectively, at 10 mmol/l. Stock solutions were stored at −20 °C. Fresh solutions (pH-adjusted) of methyl pyruvate, OAA, 3-OMG (an agonist of TXNIP), d-allose (an agonist of TXNIP) and recombinant insulin (Sigma-Aldrich) were prepared for each experiment. DMS was obtained from Acros Organics, and fresh solutions (pH-adjusted) were prepared before each experiment. For competitive-growth assays, 5 mmol/l methyl pyruvate, 5 mmol/l dimethyl succinate (DMS) and 5 mmol/l OAA were used. The CNR2 agonist HU308 was obtained from Cayman Chemical. To avoid inflammation-related effects in mice, bone marrow cells were extracted from mice (Supplementary Table 4) younger than 6 weeks of age without signs of inflammation. All mouse experiments were conducted in compliance with institutional approval by the University of California, San Francisco Institutional Animal Care and Use Committee. Bone marrow cells were obtained by flushing cavities of femur and tibia with PBS. After filtration through a 70-μm filter and depletion of erythrocytes using a lysis buffer (BD PharmLyse, BD Biosciences), washed cells were either frozen for storage or subjected to further experiments. Bone marrow cells were cultured in IMDM (Invitrogen) with GlutaMAX containing 20% fetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin and 50 μM β-mercaptoethanol. To generate pre-B ALL (Ph+ ALL-like) cells, bone marrow cells were cultured in 10 ng/ml recombinant mouse IL-7 (PeproTech) and retrovirally transformed by BCR–ABL1. BCR–ABL1-transformed pre-B ALL cells were propagated only for short periods of time and usually not for longer than 2 months to avoid acquisition of additional genetic lesions during long-term cell culture. To generate myeloid leukaemia (CML-like) cells, the myeloid-restricted protocol described previously30 was used. Bone marrow cells were cultured in 10 ng/ml recombinant mouse IL-3, 25 ng/ml recombinant mouse IL-6, and 50 ng/ml recombinant mouse SCF (PeproTech) and retrovirally transformed by BCR–ABL1. Immunophenotypic characterization was performed by flow cytometry. For conditional deletion, a 4-OHT-inducible, Cre-mediated deletion system was used. For retroviral constructs used, see Supplementary Table 5. Transfection of retroviral constructs (Supplementary Table 5) was performed using Lipofectamine 2000 (Invitrogen) with Opti-MEM medium (Invitrogen). Retroviral supernatant was produced by co-transfecting HEK 293FT cells with the plasmids pHIT60 (gag-pol) and pHIT123 (ecotropic env). Lentiviral supernatant was produced by co-transfecting HEK 293FT cells with the plasmids pCDNL-BH and VSV-G or EM140. 293FT cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) with GlutaMAX containing 10% fetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin, 25 mmol/l HEPES, 1 mmol/l sodium pyruvate and 0.1 mmol/l non-essential amino acids. Regular medium was replaced after 16 h by growth medium containing 10 mmol /l sodium butyrate. After incubation for 8 h, the medium was changed back to regular growth medium. After 24 h, retroviral supernatant was collected, filtered through a 0.45-μm filter and loaded by centrifugation (2,000g, 90 min at 32 °C) onto 50 μg/ml RetroNectin- (Takara) coated non-tissue 6-well plates. Lentiviral supernatant produced with VSV-G was concentrated using Lenti-X Concentrator (Clontech), loaded onto RetroNectin-coated plates and incubated for 15 min at room temperature. Lentiviral supernatant produced with EM140 was collected, loaded onto RetroNectin-coated plates and incubated for 30 min at room temperature. Per well, 2–3 × 106 cells were transduced by centrifugation at 600g for 30 min and maintained for 48 h at 37 °C with 5% CO before transferring into culture flasks. For cells transduced with lentiviral supernatant produced with EM140, supernatant was removed the day after transduction and replaced with fresh culture medium. Cells transduced with oestrogen-receptor fusion proteins were induced with 4-OHT (1 μmol/l). Cells transduced with constructs carrying an antibiotic-resistance marker were selected with its respective antibiotic. For loss-of-function studies, dominant-negative variants of IKZF1 (DN-IKZF1, lacking the IKZF1 zinc fingers 1–4) and PAX5 (DN-PAX5; PAX5–ETV6 fusion) were cloned from patient samples. Expression of DN-IKZF1 was induced by doxycycline (1 μg/ml), while activation of DN-PAX5 was induced by 4-OHT (1 μg/ml) in patient-derived pre-B ALL cells carrying IKZF1 and PAX5 wild-type alleles, respectively. Inducible reconstitution of wild-type IKZF1 and PAX5 in haploinsufficient pre-B ALL cells carrying deletions of either IKZF1 (IKZF1∆) or PAX5 (PAX5∆) were also studied. Lentiviral constructs used are listed in Supplementary Table 5. A doxycycline-inducible TetOn vector system was used for inducible expression of PAX5 in mouse BCR–ABL1 pre-B ALL. The retroviral constructs used are listed in Supplementary Table 5. To study the effects of B-cell- versus myeloid-lineage identity in genetically identical mouse leukaemia cells, a doxycycline-inducible TetOn-CEBPα vector system31 was used to reprogram B cells. Mouse BCR–ABL1 pre-B ALL cells expressing doxycycline-inducible CEBPα or an empty vector were induced with doxycycline (1 μg/ml). Conversion from the B-cell lineage (CD19+Mac1−) to the myeloid lineage (CD19−Mac1+) was monitored by flow cytometry. For western blots, B-lineage cells (CD19+Mac1−) and CEBPα-reprogrammed cells (CD19−Mac1+) were sorted from cells expressing an empty vector or CEBPα, respectively, following doxycycline induction. For metabolic assays, sorted B-lineage cells and CEBPα-reprogrammed cells were cultured (with doxycycline) for 2 days following sorting, and were then seeded in fresh medium for measurement of glucose consumption (normalized to cell numbers) and total ATP levels (normalized to total protein). To study Lkb1 deletion in the context of CEBPα-mediated reprogramming, BCR–ABL1-transformed Lkb1fl/fl pre-B ALL cells expressing doxycycline-inducible CEBPα were transduced with 4-OHT(1 μg/ml) inducible Cre-GFP (Cre-ERT2-GFP). Without sorting for GFP+ cells, cells were induced with doxycycline and 4-OHT. Viability (expressed as relative change of GFP+ cells) was measured separately in B-lineage (gated on CD19+ Mac1−) and myeloid lineage (gated on CD19− Mac1+) populations. To study whether Lkb1 deletion causes CEBPα-dependent effects on metabolism and signalling, Lkb1fl/fl BCR–ABL1 B-lineage ALL cells expressing doxycycline-inducible CEBPα or an empty vector were transduced with 4-OHT-inducible Cre-GFP. After sorting for GFP+ populations, cells were induced with doxycycline. B-lineage cells (CD19+ Mac1−) and CEBPα-reprogrammed cells (CD19− Mac1+) were sorted from cells expressing an empty vector or CEBPα, respectively. Sorted cells were cultured with doxycycline and induced with 4-OHT. Protein lysates were collected on day 2 following 4-OHT induction. For metabolomics, sorted cells were re-seeded in fresh medium on day 2 following 4-OHT induction and collected for metabolite extraction. For CRISPR/Cas9-mediated deletion of target genes, all constructs including lentiviral vectors expressing gRNA and Cas9 nuclease were purchased from Transomic Technologies (Supplementary Table 5; see Supplementary Table 6 for gRNA sequences). In brief, patient-derived pre-B ALL cells transduced with GFP-tagged, 4-OHT-inducible PAX5 or an empty vector were transduced with pCLIP-hCMV-Cas9-Nuclease-Blast. Blasticidin-resistant cells were subsequently transduced with pCLIP-hCMV-gRNA-RFP. Non-targeting gRNA was used as control. Constructs including lentiviral vectors expressing gRNA and dCas9-VPR used for CRISPR/dCas9-mediated activation of gene expression are listed in Supplementary Table 5. Nuclease-null Cas9 (dCas9) fused with VP64-p65-Rta (VPR) was cloned from SP-dCas9-VPR (a gift from G. Church; Addgene plasmid #63798) and then subcloned into pCL6 vector with a blasticidin-resistant marker. gRNA sequences (Supplementary Table 6) targeting the transcriptional start site of each specific gene were obtained from public databases (http://sam.genome-engineering.org/ and http://www.genscript.com/gRNA-database.html)32. gBlocks Gene Fragments were used to generate single-guide RNAs (sgRNAs) and were purchased from Integrated DNA Technologies, Inc. Each gRNA was subcloned into pCL6 vector with a dsRed reporter. Patient-derived pre-B ALL cells transduced with either GFP-tagged inducible PAX5 or an empty vector were transduced with pCL6-hCMV-dCas9-VPR-Blast. Blasticidin-resistant cells were used for subsequent transduction with pCL6-hCMV-gRNA-dsRed, and dsRed+ cells were further analysed by flow cytometry. For each target gene, 2–3 sgRNA clones were pooled together to generate lentiviruses. Non-targeting gRNA was used as control. To elucidate the mechanistic contribution of PAX5 targets, the percentage of GFP+ cells carrying gRNA(s) for each target gene was monitored by flow cytometry upon inducible activation of GFP-tagged PAX5 or an empty vector in patient-derived pre-B ALL cells in competitive-growth assays. Cells were lysed in CelLytic buffer (Sigma-Aldrich) supplemented with a 1% protease inhibitor cocktail (Thermo Fisher Scientific). A total of 20 μg of protein mixture per sample was separated on NuPAGE (Invitrogen) 4–12% Bis-Tris gradient gels or 4–20% Mini-PROTEAN TGX precast gels, and transferred onto nitrocellulose membranes (Bio-Rad). The primary antibodies used are listed in Supplementary Table 7. For protein detection, the WesternBreeze Immunodetection System (Invitrogen) was used, and light emission was detected by either film exposure or the BioSpectrum Imaging system (UPV). Approximately 106 cells per sample were resuspended in PBS blocked using Fc blocker for 10 min on ice, followed by staining with the appropriate dilution of the antibodies or their respective isotype controls for 15 min on ice. Cells were washed and resuspended in PBS with propidium iodide (0.2 μg/ml) or DAPI (0.75 μg/ml) as a dead-cell marker. The antibodies used for flow cytometry are listed in Supplementary Table 7. For competitive-growth assays, the percentage of GFP+ cells was monitored by flow cytometry. For annexin V staining, annexin V binding buffer (BD Bioscience) was used instead of PBS and 7-aminoactinomycin D (7AAD; BD Bioscience) instead of propidium iodide. Phycoerythrin-labelled annexin V was purchased from BD Bioscience. For BrdU staining, the BrdU Flow Kit was purchased from BD Bioscience and used according to the manufacturer’s protocol. Methylcellulose colony-forming assays were performed with 10,000 BCR–ABL1 pre-B ALL cells. Cells were resuspended in mouse MethoCult medium (StemCell Technologies) and cultured on 3-cm dishes, with an extra water supply dish to prevent evaporation. Images were taken and colony numbers were counted after 14 days. Cell viability upon the genetic loss of function of target genes and/or inducible expression of PAX5 was monitored by flow cytometry using propidium iodide (0.2 μg/ml) as a dead-cell marker. To study the effects of an AMPK inhibitor (BML275), glucocorticoids (dexamethasone and prednisolone), CNR2 agonist (HU308), or TXNIP agonists (3-OMG and d-allose), 40,000 human or mouse leukaemia cells were seeded in a volume of 80 μl in complete growth medium on opaque-walled, white 96-well plates (BD Biosciences). Compounds were added at the indicated concentrations giving a total volume of 100 μl per well. After culturing for 3 days, cells were subjected to CellTiter-Glo Luminescent Cell Viability Assay (Promega). Relative viability was calculated using baseline values of cells treated with vehicle control as a reference. Combination index (CI) was calculated using the CalcuSyn software to determine interaction (synergistic, CI < 1; additive, CI = 1; or antagonistic, CI > 1) between the two agents. Constant ratio combination design was used. Concentrations of BML275, d-allose, 3-OMG and HU308 used are indicated in the figures. Concentrations of Dex used were tenfold lower than those of BML275. Concentrations of prednisolone used were twofold lower than those of BML275. To determine the number of viable cells, the trypan blue exclusion method was applied, using the Vi-CELL Cell Counter (Beckman Coulter). ChIP was performed as described previously33. Chromatin from fixed patient-derived Ph+ ALL cells (ICN1) was isolated and sonicated to 100–500-bp DNA fragments. Chromatin fragments were immunoprecipitated with either IgG (as a control) or anti-Pax5 antibody (see Supplementary Table 7). Following reversal of crosslinking by formaldehyde, specific DNA sequences were analysed by quantitative real-time PCR (see Supplementary Table 8 for primers). Primers were designed according to ChIP–seq tracks for PAX5 antibodies in B lymphocytes (ENCODE, Encyclopedia of DNA Elements, GM12878). ChIP–seq tracks for PAX5, IKZF1, EBF1 and TCF3 antibodies in a normal B-cell sample (ENCODE GM12878, UCSC genome browser) on INSR, GLUT1, GLUT3, GLUT6, HK2, G6PD, NR3C1, TXNIP, CNR2 and LKB1 gene promoter regions are shown. CD19 and ACTA1 served as a positive and a negative control gene, respectively. The y axis represents the normalized number of reads per million reads for peak summit for each track. The ChIP–seq peaks were called by the MACS peak-caller by comparing read density in the ChIP experiment relative to the input chromatin control reads, and are shown as bars under each wiggle track. Gene models are shown in UCSC genome browser hg19. Extracellular glucose levels were measured using the Amplex Red Glucose/Glucose Oxidase Assay Kit (Invitrogen), according to the manufacturer’s protocol. Glucose concentrations were measured in fresh and spent medium. Total ATP levels were measured using the ATP Bioluminescence Assay Kit CLS II (Roche) according to the manufacturer’s protocol. In fresh medium, 1 × 106 cells per ml were seeded and treated as indicated in the figure legends. Relative levels of glucose consumed and total ATP are shown. All values were normalized to cell numbers (Figs 1b, c, 2c (glucose uptake), 3a and Extended Data Figs 2c, 4f, 6d) or total protein (Fig. 2c, ATP levels). Numbers of viable cells were determined by applying trypan blue dye exclusion, using the Vi-CELL Cell Counter (Beckman Coulter). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse XFe24 Flux Analyzer with an XF Cell Mito Stress Test Kit and XF Glycolysis Stress Test Kit (Seahorse Bioscience) according to the manufacturer’s instructions. All compounds and materials were obtained from Seahorse Bioscience. In brief, 1.5 × 105 cells per well were plated using Cell-Tak (BD Biosciences). Following incubation in XF-Base medium supplemented with glucose and GlutaMAX for 1 h at 37 °C (non-CO incubator) for pH stabilization, OCR was measured at the resting stage (basal respiration in XF Base medium supplemented with GlutaMax and glucose) and in response to oligomycin (1 μmol/l; mitochondrial ATP production), mitochondrial uncoupler FCCP (5 μmol/l; maximal respiration), and respiratory chain inhibitor antimycin and rotenone (1 μmol/l). Spare respiratory capacity is the difference between maximal respiration and basal respiration. ECAR was measured under specific conditions to generate glycolytic profiles. Following incubation in glucose-free XF Base medium supplemented with GlutaMAX for 1 h at 37 °C (non-CO incubator) for pH stabilization, basal ECAR was measured. Following measurement of the glucose-deprived, basal ECAR, changes in ECAR upon the sequential addition of glucose (10 mmol/l; glycolysis), oligomycin (1 μmol/l; glycolytic capacity), and 2-deoxyglucose (0.1 mol/l) were measured. Glycolytic reserve was determined as the difference between oligomycin-stimulated glycolytic capacity and glucose-stimulated glycolysis. All values were normalized to cell numbers (Extended Data Fig. 2c) or total protein (Extended Data Figs 3a, 7a, b 8f) and are shown as the fold change relative to basal ECAR or OCR. Metabolite extraction and mass-spectrometry-based analysis were performed as described previously34. Metabolites were extracted from 2 × 105 cells per sample using the methanol/water/chloroform method. After incubation at 37 °C for the indicated time, cells were rinsed with 150 mM ammonium acetate (pH 7.3), and 400 μl cold 100% methanol (Optima* LC/MS, Fisher) and then 400 μl cold water (HPLC-Grade, Fisher) was added to cells. A total of 10 nmol norvaline (Sigma) was added as internal control, followed by 400 μl cold chloroform (HPLC-Grade, Fisher). Samples were vortexed three times over 15 min and spun down at top speed for 5 min at 4 °C. The top layer (aqueous phase) was transferred to a new Eppendorf tube, and samples were dried on Vacufuge Plus (Eppendorf) at 30 °C. Extracted metabolites were stored at −80 °C. For mass spectrometry-based analysis, the metabolites were resuspended in 70% acetonitrile and 5 μl used for analysis with a mass spectrometer. The mass spectrometer (Q Exactive, Thermo Scientific) was coupled to an UltiMate3000 RSLCnano HPLC. The chromatography was performed with 5 mM NH AcO (pH 9.9) and acetonitrile at a flow rate of 300 μl/min starting at 85% acetonitrile, going to 5% acetonitrile at 18 min, followed by an isocratic step to 27 min and re-equilibration to 34 min. The separation was achieved on a Luna 3u NH2 100A (150 × 2 mm) (Phenomenex). The Q Exactive was run in polarity switching mode (+3 kV/−2.25 kV). Metabolites were detected based on retention time (t ) and on accurate mass (± 3 p.p.m.). Metabolite quantification was performed as area-under-the-curve (AUC) with TraceFinder 3.1 (Thermo Scientific). Data analysis was performed in R (https://www.r-project.org/), and data were normalized to the number of cells. Relative amounts were log -transformed, median-centred and are shown as a heat map. To generate a model for pre-leukaemic B cell precursors expressing BCR–ABL1, BCR–ABL1 knock-in mice were crossed with Mb1-Cre deleter strain (Mb1-Cre; Bcr+/LSL-BCR/ABL) for excision of a stop-cassette in early pre-B cells. Bone marrow cells collected from Mb1-Cre; Bcr+/LSL-BCR/ABL mice cultured in the presence of IL-7 were primed with vehicle control or a combination of OAA (8 mmol/l), DMS (8 mmol/l) and insulin (210 pmol/l). Following a week of priming, cells were maintained and expanded in the presence of IL-7, supplemented with vehicle control or a combination of OAA (0.8 mmol/l) and DMS (0.8 mmol/l) for 4 weeks. Pre-B cells from Mb1-Cre; Bcr+/LSL-BCR/ABL mice expressed low levels of BCR–ABL1 tagged to GFP, and were analysed by flow cytometry for surface expression of GFP and CD19. The methylcellulose colony-forming assays were performed with 10,000 cells treated with vehicle control or metabolites. Cells were resuspended in mouse MethoCult medium (StemCell Technologies) and cultured on 3-cm diameter dishes, with an extra water supply dish to prevent evaporation. Images were taken and colony numbers counted after 14 days. For in vivo transplantation experiments, cells were treated with vehicle control or metabolites (OAA/DMS) for 6 weeks. One million cells were intravenously injected into sublethally irradiated (250 cGy) 6–8-week-old female NSG mice (n = 7 per group). Mice were randomly allocated into each group, and the minimal number of mice in each group was calculated by using the ‘cpower’ function in R/Hmisc package. No blinding was used. Each mouse was killed when it became terminally sick and showed signs of leukaemia burden (hunched back, weight loss and inability to move). The bone marrow and spleen were collected for flow cytometry analyses for leukaemia infiltration (CD19, B220). After 63 days, all remaining mice were killed and bone marrow and spleens from all mice were analysed by flow cytometry. Statistical analysis was performed using the Mantel–Cox log-rank test. All mouse experiments were in compliance with institutional approval by the University of California, San Francisco Institutional Animal Care and Use Committee. Following cytokine-independent proliferation, BCR–ABL1-transformed Lkb1fl/fl or AMPKa2fl/fl pre-B ALL cells were transduced with 4-OHT-inducible Cre or an empty vector control. For ex vivo deletion, deletion was induced 24 h before injection. For in vivo deletion, deletion was induced by 4-OHT (0.4 mg per mouse; intraperitoneal injection). Approximately 106 cells were injected into each sublethally irradiated (250 cGy) NOD/SCID mouse. Seven mice per group were injected via the tail vein. We randomly allocated 6–8-week-old female NOD/SCID or NSG mice into each group. The minimal number of mice in each group was calculated using the ‘cpower’ function in R/Hmisc package. No blinding was used. When a mouse became terminally sick and showed signs of leukaemia burden (hunched back, weight loss and inability to move), it was killed and the bone marrow and/or spleen were collected for flow cytometry analyses for leukaemia infiltration. Statistical analysis was performed by Mantel–Cox log-rank test. In vivo expansion and leukaemia burden were monitored by luciferase bioimaging. Bioimaging of leukaemia progression in mice was performed at the indicated time points using an in vivo IVIS 100 bioluminescence/optical imaging system (Xenogen). d-luciferin (Promega) dissolved in PBS was injected intraperitoneally at a dose of 2.5 mg per mouse 15 min before measuring the luminescence signal. General anaesthesia was induced with 5% isoflurane and continued during the procedure with 2% isoflurane introduced through a nose cone. All mouse experiments were in compliance with institutional approval by the University of California, San Francisco Institutional Animal Care and Use Committee. Data are shown as mean ± s.d. unless stated. Statistical significance was analysed by using Grahpad Prism software or R software (https://www.r-project.org/) by using two-tailed t-test, two-way ANOVA, or log-rank test as indicated in figure legends. Significance was considered at P < 0.05. For in vitro experiments, no statistical methods were used to predetermine the sample size. For in vivo transplantation experiments, the minimal number of mice in each group was calculated through use of the ‘cpower’ function in the R/Hmisc package. No animals were excluded. Overall survival and relapse-free survival data were obtained from GEO accession number GSE11877 (refs 35, 36) and TCGA. Kaplan–Meier survival analysis was used to estimate overall survival and relapse-free survival. Patients with high risk pre-B ALL (COG clinical trial, P9906, n = 207; Supplementary Table 10) were segregated into two groups on the basis of high or low mRNA levels with respect to the median mRNA values of the probe sets for the gene of interest. A log-rank test was used to compare survival differences between patient groups. R package ‘survival’ Version 2.35-8 was used for the survival analysis and Cox proportional hazards regression model in R package for the multivariate analysis (https://www.r-project.org/). The investigators were not blinded to allocation during experiments and outcome assessment. Experiments were repeated to ensure reproducibility of the observations. Gel scans are provided in Supplementary Fig. 1. Gene expression data were obtained from the GEO database accession numbers GSE32330 (ref. 12), GSE52870 (ref. 37), and GSE38463 (ref. 38). Patient-outcome data were derived from the National Cancer Institute TARGET Data Matrix of the Children’s Oncology Group (COG) Clinical Trial P9906 (GSE11877)35, 36 and from TCGA (the Cancer Genome Atlas). GEO accession details are provided in Supplementary Tables 9 and 10. ChIP–seq tracks for PAX5, IKZF1, EBF1 and TCF3 antibodies in a normal B-cell sample (ENCODE GM12878, UCSC genome browser) on INSR, GLUT1, GLUT3, GLUT6, HK2, G6PD, NR3C1, TXNIP, CNR2 and LKB1 gene promoter regions are shown in UCSC genome browser hg19. All other data are available from the corresponding author upon reasonable request.
Javaid S.,Harvard University |
Zhang J.,Harvard University |
Zhang J.,Roswell Park Memorial Institute |
Zhang J.,Xi'an Jiaotong University |
And 15 more authors.
Cell Reports | Year: 2013
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.
News Article | November 18, 2015
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.
Sun L.,State University of New York at Stony Brook |
Veith J.M.,Roswell Park Memorial Institute |
Pera P.,Roswell Park Memorial Institute |
Bernacki R.J.,Roswell Park Memorial Institute |
Ojima I.,State University of New York at Stony Brook
Bioorganic and Medicinal Chemistry | Year: 2010
Novel paclitaxel-mimicking alkaloids were designed and synthesized based on a bioactive conformation of paclitaxel, that is, REDOR-Taxol. The alkaloid 2 bearing a 5-7-6 tricyclic scaffold mimics REDOR-Taxol best among the compounds designed and was found to be the most potent compound against several drug-sensitive and drug-resistant human cancer cell lines. MD simulation study on the paclitaxel mimics 1 and 2 as well as REDOR-Taxol bound to the 1JFF tubulin structure was quite informative to evaluate the level of mimicking. The MD simulation study clearly distinguishes the 5-6-6 and 5-7-6 tricyclic scaffolds, and also shows substantial difference in the conformational stability of the tubulin-bound structures between 2 and REDOR-Taxol. The latter may account for the large difference in potency, and provides critical information for possible improvement in the future design of paclitaxel mimics. © 2010 Elsevier Ltd. All rights reserved.
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 | Year: 2015
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.
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 | Year: 2015
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.