Germ-line or somatic inactivation of BRCA1 is a defining feature for a portion of human breast cancers. Here we evaluated the anti-proliferative activity of 198 FDA-approved and experimental drugs against four BRCA1-mutant  and four BRCA1-wild-type  breast cancer cell lines. We found that all BRCA1-mutant cell lines were insensitive to inhibitors of mitogen-activated protein kinase kinase 1 and 2 (MEK1/2) Selumetinib and Pimasertib in contrast to BRCA1-wildtype control cell lines. However, unexpectedly, only two BRCA1-mutant cell lines, HCC1937 and MDA-MB-436, were hypersensitive to a nucleotide analogue 6-thioguanine (6-TG). SUM149PT cells readily formed radiation-induced RAD51-positive nuclear foci indicating a functional homologous recombination, which may explain their resistance to 6-TG. However, the reason underlying 6-TG resistance of SUM1315MO2 cells remains unclear. Our data reveal a remarkable heterogeneity among BRCA1-mutant cell lines and provide a reference for future studies. Germ-line mutations in BRCA1 are associated with about 25% of familial breast and ovarian cancers, while somatic inactivation of BRCA1 is observed in up to 5% of sporadic breast cancers14789BRCA1-deficient breast cancers are four to eight time less likely to express estrogen alpha receptor (ERα) compared with sporadic breast cancers of similar grade, and tend to lack expression of progesterone, and ErbB2 receptors, that formally defines them as triple-negative tumors13151718202122Here we sought to investigate common and distinct features for BRCA1-mutant cell lines in comparison with a panel of BRCA1-proficient cell lines. We tested growth-inhibiting effects of 198 FDA-approved and experimental drugs on four BRCA1-mutant and four comparable BRCA1-wild-type human breast cancer cell lines. We found that all BRCA1-mutant cell lines were relatively resistant to MEK1/2 inhibitors. In addition, two of the four BRCA1-mutant cell lines were hypersensitive to 6-thioguanine (6-TG) consistent with earlier reports predicting such effect for HR-deficient cells. We also found possible explanations for resistance of two other BRCA1-mutant cell lines to 6-TG, thus providing support to this gene-drug interaction.High-throughput small molecule screening has been widely used to identify pharmacologically-relevant compounds targeting cancer cells with specific genetic abnormalities. We used a locally available library of 198 FDA-approved drugs or targeted experimental drugs202223Surprisingly, only one class of small molecule compounds demonstrated a consistent pattern of growth inhibition effect differentiating between all BRCA1-wildtype as opposed to BRCA1-mutant cell lines. All four BRCA1-mutant cell lines were relatively resistant to four inhibitors of Mitogen-Activated Protein Kinase Kinase -selumetinib (AZD6244), trametinib (GSK1120212), refametinib (RDEA119) and pimasertib (AS-703026) . This reMEK is an important kinase within the ERK mitogenic signaling pathway2930Recently, 6-thioguanine (6-TG) was found to selectively kill BRCA2-deficient cells due to its essential role in HR, which is required during mismatch repairFirst, to explain this result, we re-evaluated the expression of BRCA1 and the HR proficiency of all cell lines. Western blot analysis did not reveal a full-length BRCA1 protein in BRCA1-mutated cell lines in contrast to control cells . In addiAlternatively, resistance to 6-TG can be mediated by mutations in essential mismatch repair proteins MLH1, MSH2, MSH6, and PMS1, which are required to recognize mismatched nucleotide pairsBreast cancer cell lines are useful to investigate molecular mechanisms of breast cancer formation and identify potential therapeutics strategies. We performed a comprehensive testing of a panel of BRCA1-mutant and comparable BRCA1-wild-type breast cancer cell lines for sensitivity to 198 approved and investigational drugs to identify common vulnerabilities. Our screen revealed a remarkable heterogeneity among the four BRCA1-mutant breast cancer cell lines in their responses to most drugs. The only feature common for all four BRCA1-mutant cell lines is resistance to MEK inhibitors. On the one hand, this is consistent with an earlier finding that BRCA1-mutant breast cancer cell lines do not usually harbor activating mutations in RAS and BRAF oncogenes293050 value for isogenic BRCA1-transfected HCC1937 cells in this study is less than two-fold lower than that of the original cells, and both of them are within the micromolar range similar to our hypersensitive cell lines including HCC1937, and two orders of magnitude lower than truly 6-TG resistant cell lines. In addition, our similar screen on MDA-MB-231 cell line, which is rather sensitive to 6-TG to begin with, indicates that depletion of BRCA1 with siRNAs may further increase their sensitivity to 6-TGApart from such resistance to MEK inhibitors, BRCA1-mutant cell lines did not demonstrate any common drug vulnerabilities in our assay. Considering an earlier demonstration of sensitivity of HR-deficient cells to thiopurines, we investigated whether BRCA1 deficiency produced a similar effect. Somewhat unexpectedly, our screen revealed that two BRCA1-mutant cell lines were as resistant to 6-TG as a BRCA1-wild-type SUM229PE cell line, while two other BRCA1-mutant cell lines were almost 100-times more sensitive to 6-TG. Resistance of SUM149PT cells to 6-TG may be explained by its HR-proficiency while the reason behind SUM1315MO2 resistance remains unclear. Sensitivity of HCC1937 and MDA-MB436 cell lines to 6-TG is consistent with their HR-deficiency and mutant BRCA1 status, thus supporting the initial prediction. This result appears to contradict the study by Kinsella and colleaguesTaken together, our data provide a comprehensive description of drug vulnerabilities of existing BRCA1-mutant breast cancer cell lines. However, the output of the assay was growth inhibition, while measurement of cytotoxicity, apoptosis induction, or expression of various reporters as assay readout could produce different results2 atmosphere. The cell identity was confirmed at the FIMM technology center according to ATCC guidelines using the Promega StemElite ID System.Breast cancer cell lines HCC1937, MDA-MB-231, MDA-MB-436, MCF-7, and the non-malignant MCF10A cells were purchased from the American Type Culture Collection (ATCC). All SUM cell lines were kindly provided by Dr. John Martens . HCC1937 cells were grown in RPMI 1640, MDA-MB-231, MDA-MB-436, and MCF-7 in DMEM, supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 1 × Penicillin-Streptomycin (Life Technologies). MCF-10A cells were cultivated in DMEM/F-12 medium supplemented with 5% horse serum, 20 ng/ml EGF, 0.5 μg/ml hydrocortisone, 0.1 μg/ml cholera toxin, 10 μg/ml insulin and 1 × Penicillin-Streptomycin. SUM cell lines were all grown in Ham’s F-12 medium containing 5% FBS, 10 μg/ml insulin, 1 × Penicillin-Streptomycin (Life Technologies), and supplemented either with 10 ng/ml EGF (for SUM1315MO2), or with 0.5 μg/ml hydrocortisone (for SUM149PT and SUM229PE). Cells were propagated at 37 °C in a 5% CO2, cell viability was measured using CellTiter-Blue fluorescent assay (Promega) as described below. Dose-response curves were generated using the Studies software (Dotmatics Ltd.) as described earlier25The screening was performed essentially as described by Pemovska and colleagues4 cells/well so as to produce confluent wells after 72 hours  in a volume of 100 μl/well of corresponding media. Serial drug dilutions at desired range were added in 100 μl corresponding media. After 72 h the media was replaced with a 10% CellTiter-Blue reagent (Promega) and fluorescence at 520 nm was measured after 3 h using PHERAStar Plus plate reader (BMG Labtech) to estimate relative amounts of metabolically active cells. Dose response curves were plotted either in Excel or GraphPad Prism software.To validate the effects of individual drugs, cells were assayed in 96-well plates in at least three replicas. Cell were plated at concentrations between 0.4–1.3 × 103VO4) supplemented with a protease inhibitor tablet (Thermo Scientific). Protein concentrations were determined using the BCA Protein Assay Reagent (Pierce). Protein electrophoresis was carried out using NuPage 4–12% Bis-Tris precast gels (Invitrogen) in the MOPS running buffer according to manufacturer’s instructions, followed by blotting to a nitrocellulose membrane (Millipore). Membranes were incubated overnight at +4 °C in a blocking buffer  with the following primary antibodies: mouse-anti-BRCA1 , rabbit-anti-53BP1 , rabbit-anti-MSH2 , rabbit-anti-MLH1 , rabbit-anti-HPRT , rabbit-anti-PARP  and rabbit-anti-Beta Actin . After incubation with fluorescently labeled secondary antibodies  diluted 1:10000, membranes were scanned using a fluorescence scanner Odyssey (LI-COR Biosciences).Cells were prepared with a standard procedure using a modified RIPA buffer  for 15 min. After 3 washes with PBS++, cells were permeabilized with 0.5% Triton X-100 for 15 min. Then they were blocked for 30 min in an incubation buffer . After blocking, the cells were incubated overnight at 4 °C with mouse-anti-phospho-H2A.X  and rabbit-anti-RAD51 , or mouse-anti-BRCA1  and rabbit-anti-53BP1  antibodies. Secondary goat-anti-mouse Alexa488 and goat-anti-rabbit Alexa594 antibodies were applied for 1 hour at room temperature after 3 washes with the incubation buffer. Images were taken using Nikon Eclipse 90i fluorescent microscope and processed with the Nikon NIS-Elements AR software.To visualize DNA repair complexes, cells grown on glass coverslips for 36 hours were gamma-irradiated (5 Gy). Six hours later, cells were fixed with 2% paraformaldehyde diluted in PBS containing 1 mM CaClProteome profiling was performed using Human Phospho-Kinase Antibody Array Kit (R&D Systems) according to manufacturer’s instructions. Briefly, cells were grown in 10-cm culture dishes, rinsed with PBS, and lysed. Protein concentration was estimated with BCA Protein Assay Reagent (Pierce) and 300 μg of each cell lysate was added to pre-blocked antibody array membranes for incubation. Membranes were treated with detection antibody cocktail followed by streptavidin-HRP as instructed, and signal was detected using the enhanced chemiluminescence method .How to cite this article: Gu, Y. et al. BRCA1-deficient breast cancer cell lines are resistant to MEK inhibitors and show distinct sensitivities to 6-thioguanine. Sci. Rep. 6, 28217; doi: 10.1038/srep28217 (2016).


DNA topoisomerase I (Top1) is a DNA unwinding protein and the specific target of the camptothecin class of chemotherapeutic drugs. One of these, irinotecan, acting through its active metabolite SN-38, is used in the treatment of metastatic colorectal cancer. However, resistance to irinotecan represents a major clinical problem. Since molecular alterations in Top1 may result in resistance to irinotecan, we characterized Top1 in three human colon cancer cell lines with acquired resistance to SN-38.TOP1 gene copy number and gene sequence, Top1 expression (mRNA and protein), Top1 enzymatic activity in the absence and presence of drug, and Top1-DNA cleavage complexes in drug treated cells. TOP1 mutations were validated by PCR using mutant specific primers. Furthermore, cross-resistance to two indenoisoquinoline Top1-targeting drugs (NSC 725776 and NSC 743400) and two Top2-targeting drugs (epirubicin and etoposide) was investigated.Three SN-38 resistant (20–67 fold increased resistance) cell lines were generated and compared to wild-type parental cells with regards to: TOP1 gene copy number aberrations: A TOP1 gene copy gain and a loss of chromosome 20, respectively. One resistant cell line harbored a pair of yet unreported TOP1 mutations (R364K and G717R) in close proximity to the drug binding site. Mutant TOP1 was expressed at a markedly higher level than wild-type TOP1. None or very small reductions were observed in Top1 expression or Top1 activity in the absence of drug. In all three SN-38 resistant cell lines Top1 activity was maintained in the presence of high concentrations of SN-38. None or only partial cross-resistance were observed for etoposide and epirubicin, respectively. SN-38 resistant cells with wild-type TOP1 remained sensitive to NSC 743400, while cells with mutant TOP1 was fully cross-resistant to both indenoisoquinolines. Top1-DNA cleavage complex formation following drug treatment supported the other findings.Two of three SN-38 resistant cell lines carried TOP1 mutations were identified, and it was underlined that cross-resistance to the new indenoisoquinoline drugs depends on the specific underlying molecular mechanism of resistance to SN-38.This study adds to the growing knowledge about resistance mechanisms for Top1-targeting chemotherapeutic drugs. Importantly, two yet unreported  In metastatic colorectal cancer (mCRC), current chemotherapeutic treatment options consist of either 5-fluorouracil and folinic acid with oxaliplatin (FOLFOX) or 5-fluorouracil and folinic acid with irinotecan (FOLFIRI) , 2. HoweTOP1 located on the long arm (q) of chromosome 20. Top1 binds supercoiled DNA, nicks a DNA strand allowing its rotation around the intact strand, and then religates the DNA . Samples were sonicated (15 s), kept for 5 min on ice and heated for 5 min at 95 °C. Total protein was determined using the Lowry method, i.e. absorbance measurement and using a standard curve of bovine serum albumin in sample buffer. Samples were diluted and 10 μg of total protein per sample were applied on a 4–20 % gradient gel  together with a marker , and subjected to electrophoresis . Gel was transferred to a polyvinylidene difluoride (PVDF) membrane by semi-dry blotting 5 V overnight. Membrane was blocked for 1 h at room temperature in 5 % milk in PBS-Tween20 buffer. Subsequently, the membrane was stained with primary antibody, either anti-Top1  or anti-actin  (loading control), by incubation overnight at 4 °C. Membrane was washed three times, and incubated with HRP-conjugated secondary antibody  for 1 h at 30 °C. Subsequently, the membrane was washed three times and substrate  was added according to the manufacturer’s instructions. Signal was developed in the dark room using a photographic film. Western blots were replicated.Cells were trypsinized, resuspended and washed in cold PBS and pellet frozen at -80 °C. The Top1 ELISA was performed as previously described . Cell peCells were trypsinized, resuspended and counted, and for each cell line 1 million cells were pipetted to each of three eppendorf tubes on ice. Cells were pelleted  and snap-frozen in dry ice and ethanol and stored at -80 °C until analysis. Nuclear extracts were prepared essentially as previously described , and Top14C]thymidine and chased with radioisotope-free medium 4 h before drug treatment. Cells were treated with either SN-38  or NSC 743400 (1 μM) for 1 h. Untreated cells were included as controls. Cells were harvested by scraping and quickly pipetted to tubes on ice. Cell aliquots were placed in ice-cold PBS and irradiated with 3000 rad to break the DNA. Cells were layered onto polyvinylchloride-acrylic copolymer (protein adsorbing) filters and lysed with LS-10 . DNA was eluted from the filters with tetrapropylammonium hydroxide-EDTA (pH 12.15). After elution, filters were incubated for 1 h at 65 °C with 1 M HCl and an additional hour at room temperature in the presence of 0.4 M NaOH. Radioactivity in fractions and filters was measured with a liquid scintillation analyzer  and the fraction of DNA retained on the filter at each time point was calculated. The results were converted to rad-equivalent (a measure of DNA-protein crosslinks). Experiments were done twice each with two technical replicates.Top1-DNA cleavage complexes (DPCs) were detected using alkaline elution as previously described . In briep-value less than 0.05 was considered significant.mRNA expression values in triplicate was compared between corresponding parental and resistant cell lines using unpaired Student’s t-tests. Formation of DNA crosslinks in different cell lines following drug treatment  was compared using paired Student’s t-tests. A www.ebi.ac.uk).The evolutionary conservation of selected amino acids in the Top1 protein was analyzed across seven different species  using multiple sequence alignment , in both the parental and resistant cells  or mutant (mt) sequences at amino acid residue positions 364 and 717 in TOP1. A fragment of TOP1 was amplified by PCR using combinations of primers and cDNA (made from mRNA) from parental or resistant cells , while no fragment was amplified using the combination of mutant primers. A relatively weak band was seen using 364wt-717mt primers, which might represent unspecific primer binding or a weak expression of mutant G717R TOP1 in parental cells. On the other hand, SN-38-resistant HCT116-SN38 cells only weakly expressed wild-type TOP1, while 364mt-717mt and the wt-mt combinations of TOP1 were highly expressed. Single-mutant (wt-mt or mt-wt combinations of primers) TOP1 appeared to be more highly expressed than double-mutant (mt-mt) TOP1.The identified see Fig. . ParentaTOP1 mRNA expression was determined by microarray analysis [analysis   assay previously described by Stougaard et al.  and AndeThe cross-resistance of the SN-38-resistant cells were determined against two indenoisoquinoline non-camptothecin Top1-targeting drugs in clinical trial; NSC 725776 (LMP776/indimitecan) and NSC 743400 (LMP400/indotecan), as well as two drugs targeting Top2; the anthracycline epirubicin and etoposide . The first was a TOP1 gene copy gain in a subpopulation of cells  independently of centromere-20, a marker for the chromosome bearing TOP1 (HCT116-SN38), while the other was a loss of one copy of chromosome 20 (including one copy of TOP1) (HT29-SN38). Two cell line studies by McLeod and Keith [TOP1 gene copy number and sensitivity to SN-38 and between gene copy number and protein expression, respectively. The positive association between TOP1 amplification and expression of Top1 mRNA and protein was confirmed by another study [Looking at the nd Keith  and Romend Keith  demonstrer study , and Toper study .TOP1 we did a mutational analysis of the gene in the SN-38 resistant and parental cells. Two of three resistant cell lines did not harbor any mutations, while the third (HCT116-SN38) harbored two mutations. Both identified mutations were non-synonymous and heterozygous; one located at amino acid residue position 364  in the Top1 core domain and the other at position 717  in the C-terminal domain [TOP1 wild-type and mutant specific primers, we showed by PCR that the mutated cells (HCT116-SN38) expressed mutant TOP1 mRNA at a markedly higher level than wild-type TOP1 mRNA, even though the sequencing analysis suggested heterozygosity of the mutations. TOP1 containing a single mutation (R364K or G717R) appeared to be expressed at a higher level than double-mutant TOP1, suggesting that the mutations are present on separate alleles or in separate subpopulations in the HCT116-SN38 cells, each of them conferring resistance. This is in accordance with the FISH analysis, which detected two subpopulations in the HCT116-SN38 cell line. In line with these findings, we demonstrated that Top1 activity in nuclear extract from the HCT116-SN38 cell line was largely unaffected in the presence of high doses of SN-38, which completely eliminated the Top1 activity in parental cells, while the Top1 activity in drug absence was the same in HCT116-SN38 and parental cells. Furthermore, we measured the degree of formation of Top1-DNA cleavage complexes by alkaline elution following SN-38 treatment of cells, and showed that far fewer complexes were formed in HCT116-SN38 cells compared to parental cells. These findings suggest that SN-38 binding to Top1-DNA is hindered in the mutation-harboring cells. In previous studies, identified TOP1 mutations have clustered in regions close to the structural site where Top1 binds DNA and camptothecin, i.e. the regions 361–364 (DNA minor groove), 503–533 (minor groove) and 717-729 (major groove) [TOP1 mutations in the two other SN-38 resistant cell lines (HT29-SN38 and LoVo-SN38). However, even in these cells Top1 activity was unaffected by the presence of large concentrations of SN-38, and thus other mechanisms must be responsible for this finding.Using primers covering the full coding region of l domain , 46. Botthird HCT6-SN38 ha groove) . Some mu groove) . In a st groove)  amino ac groove) . Further groove)  showed t groove) –55. The  groove) , and sev groove) –62, incl groove) . The aut groove) . Losasso groove) . Losasso groove)  investig groove) . Gongora groove) . We did TOP1 (HT29-SN38 and LoVo-SN38), were partly cross-resistant to LMP776, while showing either no or only small cross-resistance to LMP400. Studies in our laboratory [TOP1 is wild-type. This is in line with a previous study by Antony et al. [Lastly, we assessed the three SN-38 resistant cell lines for cross-resistance to two non-camptothecin Top1-targeting drugs in clinical trials (indenoisoquinolines: NSC 725776/LMP776/indimitecan and NSC 743400/LMP400/indotecan) and two clinical Top2-targeting drugs (epirubicin and etoposide). All three SN-38 resistant cell lines displayed no or very little cross-resistance to etoposide, a specific Top2 inhibitor , 13, 46,boratory  showed tboratory , 69, whiy et al. , where Ly et al. . Our expTOP1 gene aberrations were detected in two of three cell lines, and a not previously reported pair of mutations in TOP1 was identified in one cell line. The SN-38 resistant cells displayed an interesting pattern of cross-resistance to two indenoisoquinoline Top1-targeting drugs: SN-38 resistant cells with mutant TOP1 and no overexpression of drug-efflux pump ABCG2 were resistant to LMP400, while SN-38 resistant cells with wild-type TOP1 and overexpression of ABCG2 remained sensitive to LMP400. Furthermore, cross-resistance to Top2-targeting drugs was not existent or limited. Thus, this study adds to the growing knowledge about anti-cancer resistance mechanisms for camptothecins and the new class of indenoisoquinoline Top1-targeting drugs.We generated three SN-38-resistant human colon cancer cell lines and investigated Top1. We detected no difference in the expression level of Top1 and no to very little reduction in Top1 activity in the absence of drug. A markedly increased activity of Top1 in the presence of SN-38 was seen in all three resistant cell lines.


Ostreococcus tauri RCC4221, Ostreococcus mediterraneus RCC2590, Micromonas pusilla RCC299, and Bathycoccus prasinos RCC1105. Cell division rates, taken as a proxy for fitness, systematically decline over the course of the experiment in O. tauri, but not in the three other species where the MA experiments were carried out over a smaller number of generations. However, evidence of mutation accumulation in 24 MA lines arises when they are exposed to stressful conditions, such as changes in osmolarity or exposure to herbicides. The selection coefficients, estimated from the number of cell divisions/day, varies significantly between the different environmental conditions tested in MA lines, providing evidence for advantageous and deleterious effects of spontaneous mutations. This suggests a common environmental dependence of the fitness effects of mutations and allows the minimum mutation/genome/generation rates to be inferred at 0.0037 in these species.Estimates of the fitness effects of spontaneous mutations are important for understanding the adaptive potential of species. Here, we present the results of mutation accumulation experiments over 265–512 sequential generations in four species of marine unicellular green algae,  Drosophila melanogaster  an increase of the fitness variance suggests a variation in the fitness effects of mutation between environments (iii), a change in the average fitness measured might be explained by increased selection strength in harsh conditions.MA experiments also enable the relationship between the fitness effects of mutations and the environment to be explored. Knowledge about genotype–environment (GxE) interactions is essential to understand the adaptation process, because fitness effects of mutations may change with time and spatial scales. In nogaster , C. elegrevisiae , the fitutations . The natutations : (i) a cEscherichia coli :  RCC1105 , and areO. tauri RCC4221, O. mediterraneus RCC2590, M. pusilla RCC299, and B. prasinos RCC1105. All cultures are available from the Roscoff Culture Collection (http://roscoff-culture-collection.org/). The identity of each strain was confirmed by 18S rDNA sequencing and PFGE migration : igration  at the si.e., 24 control replicates.Each experiment was started with one single cell, which divided to produce the ancestral population, from which single cells were sampled to generate independent lines by one cell inoculation . For eacS. cerevisiae, D. discoideum or Chlamydomonas reinhardtii  by k and t.If we assume that cells are uniformly distributed through the medium, the number of sampled cells, TM beads were used to calculate the abundance of the cells as described by One fifth of the microtiter plate’s volume was used for Cell counting, using a FACSCanto II flow cytometer  equipped with an air-cooled laser providing 15 mW at 488 nm with the standard filter set-up. Becton Dickinson TrucountO. tauri, 21 bottlenecks over 294 d for O. mediterraneus, 21 bottlenecks over 302 d for M. pusilla, and 16 bottlenecks over 224 d for B. prasinos . G is the number of generations/day. To compare G between different MA lines over time, the relative fitness, rG (rG = MAG/controlG), was computed. The effective population size of MA lines and control line populations at each bottleneck was estimated as the harmonic mean of the population size between t = 1 to t = 14 days. Following TS.We estimated the fitness of lines from the number of divisions/day, O. mediterraneus, M. pusilla, and B. prasinos, we used MA lines that had survived from the first to the last generations in each species for further investigations in stressful conditions: nine MA lines of O. mediterraneus, seven MA lines of M. pusilla, and eight MA lines of B. prasinos. For O. mediterraneus, 24 MA lines reached the end of the experiment, of which nine were chosen randomly for practicality.Upon completion of the MA experiments in TS was estimated as specified above. This corresponds to a total of 52 wells measures for M. pusilla, 58 for B. prasinos, and 64 for O. mediterraneus. In O. tauri, the MA experiment was completed 6 months before the start of the fitness assays under stressful conditions, so fitness assays could not be performed for this species.Before starting fitness assays, we transferred MA lines in L1 medium flasks and let them grow for 2 wk to have enough cells to inoculate cultures. Fitness assays were performed in 48-well microtiter plates, with a starting population of ∼50,000 cells/well. For herbicide tolerance tests, we used Diuron at 10 μg/L and Irgarol 1051 at 1 μg/L . The numG, and the number of sequential generations, we used data from those lines that survived throughout the experiment: 21 lines for O. tauri, 24 for O. mediterraneus, eight for B. prasinos, and seven for M. pusilla. We performed an ANOVA on the control data to test whether G changed significantly between bottleneck times. The change in fitness of MA lines as a function of time was thereafter analyzed by dividing the growth rate in the MA lines by the growth rate in the control, rG, to remove the variation in the experimental set-up through time. For each line, the relationship between the relative fitness (rG) and the number of generations was tested using Pearson’s correlation.First, to investigate the relationship between fitness, TS was calculated in all conditions using GMA and Gcontrol at each condition as explained above. We used a pairwise Student’s test to detect changes between MA lines and control. The p-value was corrected for multiple testing using the Bonferroni-Holm method  .Table S1, Table S2, Table S3, and Table S4 contain fitness data of each MA line during the experiments. Table S5, Table S6, and Table S7 contain fitness data for fitness assays in herbicides and salinity gradient conditions. Control data during MA experiments are provided in Table S8, Table S9, Table S10, and Table S11.Supplemental Material, O. mediterraneus and M. pusilla and eight cells in B. prasinos and O. tauri  for all species. The fitness values of MA lines were thus divided by the mean fitness estimation of the control, rG of O. tauri MA lines per bottleneck event decreases significantly with time . Also, four independent MA lines of the 21 had an individually significant decrease of rG  (Table S4).The average O. mediterraneus, rG significantly increased in one line . This line is the only one with a significant increase in fitness. No significant increase or decrease of within-species fitness variation of rG was detected for M. pusilla (Table S1), B. prasinos (Table S2), and O. mediterraneus (Table S3).We also investigated whether the number of lines lost varied over the course of the experiments: the data are consistent with a constant line loss over the course of the experiments in all four species. However, the observed number of lines lost was higher than expected by chance for a coefficient of variation in sampling error equal or smaller to 5% . A change of variance is as expected in stressful conditions, because of the revelation of mutation effects.Both herbicides significantly decreased fitness in the control lines in all tested species when compared to those cultured without herbicide ; the herbicides reduced growth rate by 52% and 74% for TS, are shown in TS changed between the two conditions for some identical MA lines.For each species, the selection coefficients, In all, one MA line had a significantly positive selection coefficient, while two MA lines had a significantly negative selection coefficient in the two conditions.TS in at least one herbicide, whereas five lines (21%) had a significantly positive TS.In summary, out of 24 tested lines, 12 lines (50%) had a significantly negative B. prasinos. In contrast, the control lines of both M. pusilla and O. mediterraneus grew faster in the slightly lower salinity treatment (20 g/L), and O. mediterraneus also grew faster in the lowest salinity treatment (5 g/L) than in the standard conditions (35 g/L), suggesting that lower salinity is not necessarily stressful. A change in the selection coefficient of MA lines is thus not necessarily a consequence of a stress, but just due to benign changes of an environmental parameter.MA and control lines were exposed to lower  and higher  levels of salinity than the seawater of their natural environment (35 g/L). Below, we define an environment as stressful if the controls grow more slowly in this environment than in standard conditions, the magnitude of stress being estimated by the growth rate reduction. Both high and low salinities are stressful for TS in each condition with the standard conditions (35 g/L). The variance of the fitness of MA lines was significantly higher for O. mediterraneus in the higher salinity, the most stressful condition . This was also the case for B. prasinos in the two higher and lower salinities  and at 20 g/L . In contrast, we did not detect any significant change of the variance in the fitness of M. pusilla MA lines between tested conditions.Stress may be expected to increase the fitness variance. To test this, we compared the variance of O. mediterraneus, TS was systematically negative for the MA lines. In particular, the decrease of TS was the most significant in the highest salinity, which was the most stressful. B. prasinos and M. pusilla were much more variable. In B. prasinos, almost all MA lines had a significantly higher fitness than the control under stressful conditions, whereas in M. pusilla approximately half of the lines with significantly different fitness to the control had higher fitness, and half had lower fitness. Strikingly, the MA lines in B. prasinos with higher fitness under low salinity also had higher fitness in higher salinity.The three species showed contrasting patterns in terms of the direction of selection coefficient variation, estimated from the number of cell divisions/day . In O. mIn conclusion, all 24 MA lines investigated had a significant lower or higher selection coefficient than the control lines in at least one condition, in accordance with the accumulation of spontaneous mutations in each MA line and a variation in the effects of spontaneous mutations in different environments.O. tauri, most MA lines did not show any evidence of fitness decrease during the experiment. This is despite running the experiment with a low average effective population size of around eight individuals, at maximum, over 265–272 generations. Several factors might explain the absence of fitness decrease in most MA lines.Except for O. mediterraneus, seven for M. pusilla, and eight for B. prasinos. Depending on the number of generations and the genome size, the minimum mutation rate is thus 2.72−10 mutations/site/generation for O. mediterraneus , 1.75−10 for M. pusilla , and 2.52−10 for B. prasinos . These estimates are consistent with estimates in other unicellular organisms, like C. reinhardtii . Indeed, recent MA experimental studies in C. reinhardtii . However, increasing the number of sequential generations beyond 200 was not possible: in B. prasinos and M. pusilla, the MA experiments had to be stopped as a consequence of the high line loss at each bottleneck. The number of lines lost was leading to a stagnation of the total number of independent generations in the experiments. Line loss occurred at each bottleneck from the start of the experiment and there was no trend (increase or decrease) in the number of lines lost with time. There are three possible explanations for line loss.Finally, the duration of the experiment may not have been sufficient to detect the effects of deleterious mutations. A decrease of fitness was observed in Materials and Methods). The probability of line loss is thus smaller than 10−2 in all experiments . The difference in line loss rates between species could thus be the consequence of a difference in dependence of cell division to an environmental factor, lost during the reinoculation step. This environmental factor may be a metabolite produced by the culture, e.g., a phytohormone (A third hypothesis is that line loss is not the consequence of cell death but the consequence of the absence of cell division. In lab conditions, living cells usually engage in cell division at the end of the day, after light exposure, provided nutrients are available. Without bottleneck to one single cell, line loss in culture maintenance is exceptional. However, if cell division is triggered by an environmental factor produced by the culture, it may be halted as a consequence of the reinoculation step of one single cell. Consistent with this hypothesis, we observed that lost lines were transferred from significantly smaller volumes; from 2 µl on average, while maintained lines have been transferred from 4 µl, on average, for ohormone . This hiChanges in environmental conditions clearly enable the detection of substantial variation in fitness between MA lines. This is as expected if the fitness effect of mutation changed between environments. The variance between the MA lines is greater than the variance between the control lines, suggesting that some mutations, not detected in MA standard conditions, have been fixed in our MA lines. The significant variation in fitness of some MA lines may be the result of several nonmutually exclusive factors.eN, but the overall difference in fitness between the MA and control lines is not detectable under the standard MA conditions. However, such differences in fitness might be detectable in a stressful environment because the selection intensity changes (C. reinhardtii, Kraemer and coworkers also highlight the effects of stress on the amplification of deleterious mutations and their impact on fitness (First, stressful conditions might exacerbate already existing fitness differences , so the  changes . A changeN. As a consequence, these slightly deleterious mutations, which could become advantageous in a novel environment, can accumulate in the MA lines but not in the controls. They may thereby increase the fitness in some of these MA lines. In addition, both the control and MA lines have accumulated mutations that are neutral under the original conditions but deleterious under the stressful conditions, causing the fall of fitness among MA lines.Second, the fixation of mutations, particularly slightly deleterious mutations, is faster in the MA lines because they have smaller We investigated the accumulation of mutations in four marine green picoalgae. Despite a modest number of sequential generations per MA line, we found evidence for a variation in fitness effects of spontaneous mutations from benign to stressful environments. This allowed us to estimate a minimum per genome mutation rate of 0.0037.


The rise of genomic studies in Africa – not least due to projects funded under H3Africa – is associated with the development of a small number of biorepositories across Africa. For the ultimate success of these biorepositories, the creation of cell lines including those from selected H3Africa samples would be beneficial. In this paper, we map ethical challenges in the creation of cell lines.The first challenge we identified relates to the moral status of cells living in culture. There is no doubt that cells in culture are alive, and the question is how this characteristic is relevant to ethical decision-making. The second challenge relates to the fact that cells in culture are a source of cell products and mitochondrial DNA. In combination with other technologies, cells in culture could also be used to grow human tissue. Whilst on the one hand, this feature increases the potential utility of the sample and promotes science, on the other it also enables further scientific work that may not have been specifically consented to or approved. The third challenge relates to ownership over samples, particularly in cases where cell lines are created by a biobank, and in a different country than where samples were collected. Relevant questions here concern the export of samples, approval of secondary use and the acceptability of commercialisation. A fourth challenge relates to perceptions of blood and bodily integrity, which may be particularly relevant for African research participants from certain cultures or backgrounds. Finally, we discuss challenges around informed consent and ethical review.In this paper, we sought to map the myriad of ethical challenges that need to be considered prior to making cell line creation a reality in the H3Africa project. Considering the relative novelty of this practice in Africa, such challenges will need to be considered, discussed and potentially be resolved before cell line creation in Africa becomes financially feasible and sustainable. We suggest that discussions need to be undertaken between stakeholders internationally, considering the international character of the H3Africa project. We also map out avenues for empirical research. H3Africa supports individual research projects, a bioinformatics network (H3A BioNet) and a small number of African biorepositories to store  samples.aGenomic research in Africa has received significant support under a funding programme called ‘H3Africa’, where H3 stands for Humans, Heredity and Health. H3Africa  is the creation of cell lines as a value-added service. A cell line is made by taking a human body cell such as a blood cell or skin cell and changing some of its’ genetics in order to make the cell reproduce indefinitely outside of the body. Once established, the cells in the cell line will keep on reproducing in a flask as long as they are taken care of – in other words, as long as they are fed and refreshed, and have oxygen to breathe. Because the cells in culture originate from a single cell, they are genetically uniform and share the genetic makeup of the person from whom the cell was obtained.At the most basic level, the immortalisation of cells is considered as the optimal way to preserve DNA outside the human body. Once cell lines are created, they form an inexhaustible resource for DNA and, of course, other cell products too. A second advantage to the creation of cell lines, therefore, lies in that they may enable functional genomic work to be conducted. Whereas a sample collected at one moment in time may allow for only very limited functional work (e.g. RNA expression studies), a cell line allows researchers to study cellular responses to different stimuli that emulate infection or disease. In this way, it allows researchers to identify not only genetic factors involved in disease causation or prevention, but also to study the actual effect of those factors. A third advantage is that the collection of new samples is not necessary and thus may minimise the burden and cost of patient recruitment. The preservation of cells in culture allows for the application of the latest scientific techniques on these samples. An example of such a technology in development is single-cell genomics, which allows for detailed genomic and transcriptomic studies on the basis of a single cell,2.However, the creation of cell lines also raises ethical issues, not all of which are thoroughly understood. In the African context, the creation of cell lines is relatively novel. In this paper, we will map the ethical issues around the creation of cell lines and discuss those that need to be addressed before the development of African sample repositories, as discussed by the H3Africa Working Group on Ethics.The creation of cell lines became technologically feasible in the 1950s, when the first cell line that multiplied in vitro was generated using the cells of a cancer patient called Henrietta Lacks, also known as HeLa. Since tb Many of the earlier samples were not collected purposively for the creation of cell lines. More recent research, however, has witnessed the collection of African human samples specifically for cell line creation. This is the case for the 90 HapMap cell lines derived from samples obtained from Yoruba people living in Ibadan, Nigeria[c Many more cell lines may have been developed from samples obtained from African people, but these collections are thought to be scattered across laboratories and universities across the globe, and no comprehensive record exists. In the context of H3Africa studies, the expectation is that it would ultimately be beneficial to create cell lines for (selected) samples so that samples will not be limited for future research and additional resources will not be needed for renewed sample collection but this is not currently economically and technologically feasible.Researchers have created cell lines from human specimens for over five decades, and have also used this technology to create cell lines from African samples – many of which are currently preserved by the Coriell Institute in the USA. Specific examples are cell lines created for the Biaka pygmy population from the Central African Republic and the Mbuti pygmy population from the Democratic Republic of Congo. Nigeria, and for Nigeria. The latA key question relevant to a discussion of the ethical issues relating to the creation of cell lines is whether this practice distinguishes itself from other forms of sample manipulation in a way that is morally relevant. In other words, are there aspects of the creation of cell lines that raise ethical concerns in and of themselves?On one extreme, the creation of cell lines could be considered as a way of preserving genetic material, and of ensuring access to this material in the future. Proponents of this view argue that the technique minimises harm to participants by reducing the need for additional samples, whilst maximising benefit by enabling the development of a more comprehensive picture of disease, which will ultimately be of benefit to patients. From this perspective, the creation of cell lines is not morally different from the collection of a finite genetic sample.An opposing view could be that cells in culture are living: they breathe oxygen, consume nutrients, multiply and can die. If they have the ability to die, then they logically need to be considered alive as well. This is significantly different from other genomic material collection which required the collection of a finite genomic sample. When considered in this way, the creation of cell lines touches on concepts that were previously associated with, for example, deities or superheroes – immortality and the ability to live and multiply outside of the body.in vitro under artificially engineered circumstances. They have the ability to provide information on the physiological body of the person who donated them, but do not seem to have the same ability to provide information on the other aspects that make up a person – for instance, their emotional state, thoughts and lived experiences. That cells outside of the body do have meaning may be clear from a comparison with cells in semen[The latter observation – that cells in culture are alive – may be morally significant. Obviously, cell lines in a flask are not experiencing life in the same way that a living human does, and they should therefore not be treated the same as the conscious, living persons. They share with the humans from whom they came, all of the genetic material as well as the ability to live in semen. Howeverin semen. A complin semen.A second way in which cell lines are different from finite genomic samples is that they offer a source of cell products other than just nuclear DNA. Cell lines offer access not only to mitochondrial DNA, but also to transcription RNA, expressed proteins and other cell products. This is morally relevant most importantly because it significantly increases the usefulness of the donated sample. For instance, rather than just examining the ‘genetic blueprint’, which is static, cell lines could be used to study (genetic) responses to environmental stimuli, including infection with disease-causing pathogens or therapeutic drugs. This means that rather than just focusing on an individuals’ genetic makeup and analysing the influence of genetic variants on disease, the creation of cell lines allows a researcher to study disease responses at the cellular level over time, without necessarily returning to the research participant for further samples or information, and without having to subject them to potentially harmful experiments. The expectation is that using cell lines will enable researchers to discover more about disease in a shorter timeframe – which would ultimately be beneficial for patients. Where projects focus on diseases prevalent in Africa, the expectation would be that the benefits from knowledge gained would accrue to African patients and the wider population, but it may be important to include this as a specific requirement in access decisions. If this expectation is true, then the creation of cell lines would have moral significance in ultimately promoting the wellbeing of African patients.Access to mitochondrial DNA may also be relevant for consent as it provides information about group origin, but this may not specifically have been highlighted in the consent forms. A concern could be that genetic accounts of ancestry may be in conflict with traditional narratives – which was apparently the case for the Havasupai in the United States,12. In tCells grown in culture can also be used to grow other tissues, which is not possible if a finite sample is not converted to cell lines. Using novel techniques such as those developed for the creation IPSCs,5, reseaThe use of these technologies generates a number of ethical concerns. One challenge relates to informed consent – whether the patient indeed agreed to allowing such manipulations to happen on the samples. Another challenge relates to immoral or illegal applications of these methods, for instance to support the creation of artificial gametes. Although this is currently illegal in most jurisdictions across the world, it is difficult to monitor how researchers use samples in the privacy of their laboratories.In the context of the H3Africa programme, the aim is to create biorepositories across a number of countries in Africa. The vision is that samples are shipped regionally, and that cell lines are created, maintained and distributed from these regional African centres to researchers elsewhere in the world. These biorepositories would be within African institutions and access decisions would be made by an oversight committee that is primarily composed of people residing in Africa. The intention to create African biorepositories seems to be a partial response to concerns about sample export and exploitation which have been raised in the context of genomic research-16. The An additional challenge in the African context relates to traditional perceptions of blood and the body. It is well-documented that blood carries symbolic value for many Africans-26. BlooThe way in which these challenges have traditionally been dealt with is by investing considerable effort in optimising the consent procedure. One of the primary challenges in the creation of cell lines relates to obtaining informed consent for the procedure. Consent for medical research across Africa often remains focused around specific research questions, with samples obtained for use in a particular disease-based project. All of the H3Africa projects, for instance, are disease-based meaning that enrolment and therefore consent is primarily focused on the disease in question. In those cases, sample and data sharing are often of secondary importance. This is significantly different from HapMap and 1000Genomes, where recruitment specifically focused on the creation of cell lines. A decision that needs to be made in the context of H3Africa studies, therefore, is whether consent for the creation of cell lines should be integrated in the main consent form or whether specific consent for cell line creation should be obtained. Greater scrutiny of the HapMap and 1000Genomes experiences may also be required for guidance.It is clear that against the foregoing discussions, there are many challenges in obtaining informed consent for genomic studies in Africa-33. One Cell line creation raises issues that are novel for ethics committees in two ways: first, in terms of the practice itself, and second, in terms of the opportunity if offers for sample re-use. For ethics committees across the continent, the usual practice is to approve the use of samples for specific research projects. Any subsequent uses of those samples need separate approval from that same ethics committee. In the context of H3Africa projects, the proposal is that ethics committees will approve the submission of samples to the biobank, but what remains unresolved is whether those committees will permit another body (such as a sample access committee or an ethics committee associated with the biobank) to make decisions about appropriate secondary uses of samples. It is important to recognise that many participant communities across Africa are considered ‘vulnerable’ to exploitation: poor, with low research literacy and obstructed access to healthcare. In our experience, ethics committees in Africa recognise these contextual issues and take their role in protecting the interests of participants very seriously. Similarly, some ethics committees in Africa have observed that medical research in Africa has yielded little benefit for African patients, institutions and countries, and are becoming increasingly critical about sample sharing and export. ExactlyThe creation of cell lines in central biobanks in Africa raises a number of ethical challenges relating to consent, community engagement, ownership and commercialisation and harmonisation of ethics review across the continent. The absence of good regulatory guidance regarding biobanking and its technologies on the continent of Africa raises many additional concerns that also need addressing. Although in this paper, we have presented the various ethical challenges raised by the creation of cell lines as disconnected, they share considerable overlap. For instance, the absence of a good awareness of participants’ understanding of and perspectives on cell line creation raises challenges for ethics review – most evidently in the way ethics committees in African countries seek to prevent exploitation through control over secondary use decisions. Also, the historical exploitation of African people in medical research creates suspicions around blood collection and resistance to sample export. These issues, therefore, need to be understood together rather than in isolation.There is a considerable gap in knowledge about the perspectives of African research participants on the creation of cell lines for research and therefore an urgent need to conduct empirical studies to investigate these. Such studies could be conducted in the context of current H3Africa initiatives but also, for instance, could involve members of populations for which cell lines are already commercially available. We identify a number of areas that need further work before the large-scale creation of cell lines from human samples could become a reality in Africa. Most importantly what is needed is a comprehensive understanding of the views of stakeholders across the continent on these issues. Empirical research needs to focus on  research participants from diverse populations, in rural and urban settings and from different socio-economic backgrounds. It also needs to encapsulate researchers, medical professionals, ethics committee members and policy makers. Such work needs to span a number of academic disciplines in the social sciences and humanities.Second, it is necessary to clarify the implications of the current legal frameworks in African countries, or develop these where they are not existent or appropriate. This would involve legal scholars on the continent, as well as policy makers, civil society organisations and others. Furthermore, the role of appropriate governance mechanisms in ensuring that the creation of cell lines is done ethically in a way that builds trust in the research enterprise, needs to be explored. What is clear is that such governance mechanisms must ensure that cell lines are created for and used to promote the wellbeing of patients on the African continent.Thirdly, attention needs to be given to developing a governing structure that enables scientific research whilst also protecting participant interests. Such a governing structure may require the ongoing involvement of ethics committee members and researchers from across the continent, particularly with regard to access and re-use decisions. In particular, the governance structure should consider how and whether research is likely to benefit African patients and accommodate limitations to informed consent. One suggestion could be that the scientific community revisits the idea of a ‘Hippocratic Oath’ for scientists.In the context of H3Africa, the development of a policy framework to guide the sharing of research samples in biobanks is in its final stages. Also, the Consortium has initiated a process of engagement with members of ethics committees from across the continent to understand the key ethical challenges relating to the sharing of data and samples for genomics research in Africa. From these processes, it is clear that the development of an appropriate consent model to allow for the sharing of samples and the creation of cell lines is a first priority. It is also clear that these activities need to be embedded in robust and thorough community engagement activities, but questions about how community engagement can meaningfully support the creation and redistribution of cell lines require urgent attention. Within H3Africa, some work is taking place to address these issues both through empirical work and through a process of engagement of stakeholders across the continent.aSeehttp://h3africa.org/about/white-paper (accessed 12 January 2014).bSeehttp://ccr.coriell.org/Sections/BrowseCatalog/Populations.aspx?PgId=4 (accessed 13 January 2013).cSeehttp://www.1000genomes.org/about (accessed 13 January 2014).All authors are members of the H3Africa Working Group on Ethics and Regulatory Issues, and are involved in H3Africa projects. They declare that they have no competing financial interests.All authors participated in identifying the ethical challenges pertinent to the creation of cell lines. JdV drafted the manuscript, AA, JB, KL, EM, PM, OOMOB and JS reviewed and improved the manuscript. All authors with exception of JdV are listed in alphabetical order. All authors read and approved the final version of this manuscript.Manuscript was prepared by the authors as members of Working Group on Ethics of the H3Africa Consortium.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6939/15/60/prepub


Aromatase inhibitor (AI) treatment is first-line systemic treatment for the majority of postmenopausal breast cancer patients with estrogen receptor (ER)-positive primary tumor. Although many patients benefit from treatment, some will develop resistance, and models mimicking acquired resistance will be valuable tools to unravel the resistance mechanisms and to find new treatments and biomarkers. Cell culture models for acquired resistance to the three clinically relevant AIs letrozole, anastrozole and exemestane were developed by selection and expansion of colonies of MCF-7 breast cancer cells surviving long-term AI treatment under conditions where endogenous aromatase-mediated conversion of androgen to estrogen was required for growth. Four cell lines resistant to each of the AIs were established and characterized. Maintenance of ER expression and function was a general finding, but ER loss was seen in one of twelve cell lines. HER receptor expression was increased, in particular EGFR expression in letrozole-resistant cell lines. The AI-resistant cell lines had acquired ability to grow without aromatase-mediated conversion of testosterone to estradiol, but upon withdrawal of AI treatment, testosterone induced minor growth stimulation. Letrozole, exemestane and tamoxifen were able to abrogate the testosterone stimulation but could not reduce growth to below the level in standard growth medium with AI, demonstrating cross-resistance between letrozole, exemestane and tamoxifen. In contrast, fulvestrant totally blocked growth of the AI resistant cell lines both after withdrawal of AI and with AI treatment. These data show that ER is the main driver of growth of the AI-resistant cell lines and indicate ligand-independent activation of ER. Fulvestrant is an efficient treatment option for these AI-resistant breast cancer cells, and the cell lines will be useful tools to disclose the underlying molecular mechanism for resistance to the different AIs. This emphasizes the need for early identification of resistance and new treatment options for patients with resistant tumors. Clinical data demonstrating improved outcome by combining AI therapy with HER2 targeted therapy or with PI3K/AKT/mTOR inhibitors (Endocrine therapy is up-front systemic therapy for breast cancer patients with estrogen receptor (ER)-positive tumors. The antiestrogen tamoxifen has been the key endocrine agent since the 1970s and the meta-analysis after 15 years follow-up of 5 years adjuvant tamoxifen therapy revealed reduction in both recurrence risk and death –5. Consehibitors ,11 underThe estrogen supply for postmenopausal breast tumors originates from circulatory uptake and local synthesis in both tumor cells and surrounding tissue, e.g., adipocytes, fibroblasts and inflammatory cells –15. The Surrogate models with aromatase overexpressing breast cancer cells have been developed by stable introduction of aromatase cDNA ,28–30. CCYP19A1) is very complex  supplemented with 1% heat inactivated FCS (Life Technologies), 2 mM GlutaMAX™-1 (Life Technologies) and 6 ng/ml insulin . In order to obtain growth, which depends on conversion of androgen to estrogen via the endogenous aromatase enzyme, MCF-7 cells were transferred to medium with 10% newborn calf serum (NCS) (Life Technologies) and 10−7 M testosterone (Sigma-Aldrich) as described previously ] for one week, trypsinized and seeded in serial dilutions in 24-well plates. Single colonies were transferred to new wells and gradually expanded in medium with AI. After ~2–3 months, the isolated colonies gave rise to AI-resistant cell lines, which could be grown in AI-containing medium with a weekly split ratio of ~1:25. The MCF-7 cell line was authenticated in January 2014 by DNA profiling using short tandem repeat loci performed by Leibniz-Institut DSMZ  and found to be matching the genetic profile reported for the MCF-7 cell line (DSMZ ACC 115).The parental cell line for the AI-resistant cell lines was MCF-7 subline 0.5 (MCF-7/S0.5), which originates from MCF-7 cells from the Human Cell Culture Bank  that have been stepwise adapted to grow with 0.5% fetal calf serum (FCS) . The MCFeviously . AI-resi−6 M tamoxifen (Sigma-Aldrich) and 10−7 M fulvestrant  was performed in standard growth medium with the respective AI. All growth experiments were performed with four sample replicates or more and repeated at least twice with similar results.For dose-response growth experiments, MCF-7 cells were seeded in 24-well plates (Nunc) in their standard growth medium and after one day, they were switched from 1% FCS to 10% NCS. On day 2, treatment was initiated and renewed on day 5. On day 7, cell number was determined by a crystal violet colorimetric assay . One weeR-1, LetR-3, ExeR-1 and ExeR-3 grown in standard growth medium in passages between 15–30. Lysates from MCF-7 cells grown in standard growth medium with 1% FCS or grown for 21 weeks with 10% NCS + 10−7 M testosterone were used for comparison. For analysis of estrogen-regulated proteins, lysates were prepared from MCF-7 cells and from AI-resistant cell lines (withdrawn from AI and testosterone for one week) grown for five days with 10% NCS and 10% NCS supplemented with estradiol (Sigma-Aldrich), testosterone and testosterone in combination with AIs. SDS-PAGE page and immunoblotting were performed as described previously  were prepared from early passages (5 and 6) of four cell lines resistant to each of the three AIs; letrozole, anastrozole and exemestane grown in their standard growth medium and from the selected AI-resistant cell lines; Leteviously . Antibod−7 M testosterone for five days and AI-resistant cell lines were grown in their standard medium. RNA was isolated using PureLink Micro-to-Midi Total RNA Purification system (Life Technologies) and reverse transcribed to cDNA by the High-Capacity RNA-to-cDNA kit (Life Technologies). The real-time PCR analysis was performed with Power SYBR® Green PCR Master Mix (Life Technologies) using a Rotor-Gene 3000 . All experiments were conducted in accordance to the manufacturer’s recommendations. Results were calculated based on a real-time RT-PCR relative quantification strategy and presented as mean relative gene expression levels, compared to the parental MCF-7 cells. Primers and programs for CYP19A1 were as previously described by Díaz-Cruz et al  contains low amount of estrogenic activity and MCF-7 cells require estrogen supplementation to grow continuously in 10% NCS . Both es−11 M , whereas−11 M .−6 M letrozole, 10−7 M anastrozole and 10−7 M exemestane, respectively . An initial analysis for expression of ER and the ER-regulated proteins; progesterone receptor (PR-A and PR-B) and Bcl-2 as well as the HER receptors, was performed on the cells harvested after 2.5 months with the respective AI (−7 M testosterone had very low level of ER (The testosterone stimulation of MCF-7 cell growth can be completely abrogated by addition of the third-generation AIs, letrozole, anastrozole and exemestane , but aftctive AI . All butel of ER . PR-B anel of ER . Bcl-2 lR-1 and LetR-3, and two cell lines resistant to the steroidal AI exemestane, ExeR-1 and ExeR-3. Initially, we tested the stability regarding expression of ER, PR, Bcl-2 and HER receptors. −7 M testosterone. It should be mentioned that the ER content varies with the growth conditions and that ER level was low in MCF-7 cells grown with 10% NCS + testosterone as can be seen in R-1 and ExeR-1 (R-1 and ExeR-1 grown with estradiol and with testosterone as expected due to reduced stability of estradiol-bound ER. PR-A and PR-B were hardly detectable in MCF-7 cells grown with 10% NCS and when treated with exemestane and letrozole. PR-A and PR-B were expressed at low level in AI-resistant cells grown with 10% NCS and also expressed at low level in 10% NCS + testosterone and the respective AI. Bcl-2 was expressed at comparable level in control cells with 10% NCS and in cells grown with testosterone and AI (For further analyses, we selected two cell lines resistant to the non-steroidal AI letrozole, Letd ExeR-1 . The ER e and AI . These dCYP19A1 mRNA expression using real-time RT-PCR. The analysis revealed similar CYP19A1 mRNA level in MCF-7, LetR-1, LetR-3, ExeR-1 and ExeR-3 cells (Aromatase expression is highly regulated at the transcriptional level , and to -3 cells .−7 M testosterone, and letrozole and exemestane exerted a dose-dependent growth inhibition with maximum inhibition to the basal level with 10−7 M and 10−6 M AI (R-1, LetR-3, ExeR-1 and ExeR-3, respectively. A dose-dependent inhibition of the testosterone-stimulated cell growth was seen for AI-resistant cell lines treated with letrozole (MCF-7 cells grown with 10% NCS displayed ~6–10-fold stimulation with 100−6 M AI . The groetrozole  and exemetrozole . AI trea−7 M testosterone, a dose-dependent growth inhibition of MCF-7 cells was seen. However, growth was not completely arrested after five days treatment with 10−6 M tamoxifen (−9 M (−7 M testosterone and withdrawn from AI treatment for one week, tamoxifen induced a dose-dependent growth inhibition down to the level seen in 10% NCS (−7 M fulvestrant (R-1 and ExeR-1 cells were treated with 10−7 M fulvestrant for one week, split and treated for further one week with fulvestrant. After a total of 14 days of treatment with 10−7 M fulvestrant, no viable LetR-1 or ExeR-1 cells could be detected.Tamoxifen is a weak estrogen antagonist and in medium with 10% NCS + 10amoxifen  as it isamoxifen . Fulvesten (−9 M . Dose-reen (−9 M  and withen (−9 M . In AI-r 10% NCS . Fulvestvestrant . Tamoxifvestrant . To deteAdjuvant treatment with AIs postpones or abrogates development of advanced disease, and patients with advanced disease benefit from treatment. However, most patients with advanced disease will eventually progress, and some patients receiving adjuvant AIs progress during therapy. To find new treatment options and new biomarkers for resistant tumors, it is important to gain knowledge of the molecular mechanisms involved in resistance to AIs. In this report, we describe a new series of breast cancer cell lines with acquired resistance to the clinically relevant AIs letrozole, exemestane and anastrozole, which may be useful models for studies of the resistance mechanisms. The resistant cell lines were developed from MCF-7 cells grown under conditions at which the extracellular estrogen supply via serum was low and growth stimulation was mediated via conversion of testosterone to estradiol by the endogenous aromatase enzyme. Long-term treatment of MCF-7 cells with letrozole, exemestane or anastrozole appeared to inhibit growth completely, but some cells survived treatment and gave rise to outgrowth of colonies, which could be isolated and propagated continuously in presence of the respective AI. Initially, the growth rate was slow, but a gradual increase in growth rate was observed during the first 15 weeks of treatment until constant growth rate was achieved. We believe that the surviving colonies arise from cells with inherent resistance, whereas acquired changes may be responsible for the observed increase in growth rate during long-term propagation.CYP19A1 mRNA in parental MCF-7 cells and in the four tested AI-resistant cell lines does not support this mechanism of resistance.A switch from ER-driven growth to involvement of HER receptor-driven growth has been described for antiestrogen-resistant breast cancer cells –44, and Cell number in both the letrozole- and the exemestane-resistant cell lines increased 20–25 times during one week in the standard growth medium with the respective AI, and all resistant cell lines displayed a low degree of responsiveness to growth stimulation with testosterone when AI and testosterone were withdrawn for one week. The ability of the AI-resistant cell lines to grow continuously in presence of AI and also in medium with 10% NCS demonstrates that the cell lines have acquired ability to grow without estrogen stimulation, whereas the observed growth response to testosterone indicates that AI-resistant cell lines have progressed from estrogen-dependent to estrogen-responsive cell growth. Letrozole, exemestane and tamoxifen could abrogate the testosterone-mediated growth stimulation, but could not reduce growth to below the level in standard growth medium with AI. In contrast, fulvestrant inhibited growth of the AI-resistant cell lines to below the level in 10% NCS and down to the level of MCF-7 cells which were completely growth arrested by treatment with fulvestrant. To investigate whether fulvestrant completely blocked growth of AI-resistant cell lines, AI-resistant cells treated for one week with fulvestrant were trypsinized and seeded in new culture flasks, but no growth was observed, demonstrating that fulvestrant exerted complete growth arrest of AI-resistant cell lines and supporting that growth of the AI-resistant cell lines depends primarily on ER. The severe growth inhibition seen with fulvestrant treatment of AI-resistant cell lines in their standard growth medium with AI also supports that ER drives growth of the AI-resistant cell lines. The importance of ER-mediated growth and effect of fulvestrant treatment have also been found in exemestane-resistant cell lines derived from aromatase overexpressing MCF-7 cells , in MCF-vice versa has resulted in clinical benefit for 30–50% of the patients (in vitro cell culture model.Letrozole and exemestane abrogated the testosterone-mediated growth of both letrozole- and exemestane-resistant cell lines but could not arrest cell growth, demonstrating cross-resistance between the non-steroidal AI letrozole and the steroidal AI exemestane. This is in contrast to clinical studies in which sequential treatment from a non-steroidal to a steroidal AI or patients . It shoupatients , but lacpatients  and patiTamoxifen was able to abrogate the testosterone-mediated growth stimulation of the AI-resistant cell lines but could not reduce growth further, demonstrating that tamoxifen is able to inhibit the estrogen-mediated but not the presumed ligand-independent activation of ER. In line with this, we have recently found that ER is the main driver of growth of tamoxifen-resistant cell lines . The mulIn conclusion, this report presents a large series of AI-resistant breast cancer cell lines derived from MCF-7 cells, which can be used as models to unravel the molecular mechanisms for growth of breast cancer in patients who recur after an initial response to AI therapy. We found that the majority of AI-resistant cell lines maintained ER expression and function, whereas HER receptor expression was increased. The complete growth inhibition of the AI-resistant cell lines by treatment with the ER down modulator fulvestrant demonstrates that ER is the main driver of growth of AI-resistant cell lines, supporting the potential of fulvestrant therapy for AI-resistant breast cancer.


Metastasis is one of the important reasons for the poor prognosis of hepatocellular carcinoma (HCC), abnormal glycosylation plays a pivotal role in HCC metastasis. The goal of this study was to screen and validate the transcriptional profiling of glycogenes associated with HCC metastasis.2 Profiler PCR Array, and were identified by qRT-PCR in human HCC cell lines and their orthotopic xenograft tumors. Further analyses were performed with K-mean clustering, Gene Ontology (GO) and ingenuity pathways analysis (IPA). Four differentially transcribed glycogenes were validated in clinical cancer specimens by qRT-PCR.The differentially transcribed glycogenes were screened out by the Human Glycosylation RTK-mean clustering, all of the differentially transcribed glycogenes were classified in six clusters. GO analysis of the differentially transcribed glycogenes described them in biological process, subcellular location and molecular function. Furthermore, the partial regulatory network of the differentially transcribed glycogenes was acquired through the IPA. The transcription levels of galnt3, gcnt3, man1a1, mgat5b in non-metastatic and metastatic HCC clinical cancer specimens showed the same changing trends with the results in human HCC cell lines and their orthotopic xenograft tumors, and the divergent transcription levels of gcnt3 and mgat5b were statistically significant.A total of thirty-three differentially transcribed glycogenes were obtained by comparison the transcription in the metastatic human HCC cell lines  with the transcription in the non-metastatic HCC cell line Hep3B. Seven differentially transcribed glycogenes were selected to further identification in human HCC cell lines and their orthotopic xenograft tumors. According to their trends by The transcriptional profiling of glycogenes associated with HCC metastasis was obtained and validated in this study and it might provide novel drug targets and potential biological markers for HCC metastasis. Hepatocellular carcinoma (HCC), which is the sixth common neoplasm, ranks the third in cancer mortality in the world mgat3 and mgat5 were proved to be over-expressed in human HCC cell lines, knockdown of mgat3 could promote cells invasion and increase the resistance to 5-fluorouracil in vitro mgat5 in cells could inhibit invasion and increase sensitivity to 5-fluorouracil in vitro. mgat4a was vital to tumor migration and metastasis through altering the glycosylation of CD147 mgat4b, and mgat5 were increased in mice with HCC mgat5 could partially decrease cell adhesion and promote cell proliferation through RPTPκ Glycosylation, which can be found in a variety of physiological and pathological events, is one of the most important kinds of posttranslational modifications. More than 50% of proteins in nature are presumed to have undergone glycosylation K-mean clustering and GO analysis, respectively. The possible regulatory network of them was built by the IPA. Furthermore, the differently transcribed glycogenges were validated in clinical cancer specimens by PCR Array. This work might provide the novel potential drug targets and the potential biological markers of HCC.In this study, we obtained and identified the transcriptional profiling of glycogenes in human HCC cell lines with or without metastasis potential and orthotopic xenograft tumors by PCR Array and qRT-PCR. The differentially transcribed glycogenes were classified and described by Access to human tissues complied with both Chinese laws and the guidelines of the Ethics Committee and this study was approved by the Research Ethics Committee of Zhongshan Hospital and First Affiliated Hospital of Guangxi Medical University. All participants have given written informed consent.2 in air atmosphere.The Human HCC cell lines with different metastatic potentials  have the same genetic background and were established in our Liver Cancer Institute 7 Hep3B, MHCC97L, MHCC97H or HCCLM3 cell lines were injected subcutaneously into the upper left flank region of nude mice, respectively. After tumor formation, the tumors were cut into 2 mm×2 mm×2 mm sized pieces. Nude mice that were anaesthetized with pentobarbital 45 mg/kg by intraperitoneal injection after regular disinfection were dissected and then the tumor tissue lumps were implanted into livers of nude mice. The mice were slaughtered at the 35th day after tumor implantation and the orthotopic xenograft tumors were immediately placed in liquid nitrogen. All of the in vivo experiments carried out were strictly complied with the protocol which was approved by the Shanghai Medical Experimental Animal Care Committee (Permit Number: 2009-0082).The male BALB/C nude mice (5 to 6-week-old) we obtained were from Shanghai Institute of Materia Medica . The orthotopic xenograft tumor models were established as described previously Fifteen HCC patients under-going resection in 2012 in Zhongshan Hospital, Fudan University  and First Affiliated Hospital of Guangxi Medical University  were enrolled in this study, including ten patients with non-metastatic HCC and five patients with metastatic HCC. All the tissue samples collected from the patients were performed to qRT-PCR analysis. The detailed information of these fifteen patients was described in Total RNA was harvested from cultured cells, orthotopic xenograft tumors and clinical cancer specimens with TRIzol (Invitrogen) and purified with the RNeasy MinElute Cleanup Kit (Qiagen) according to the manufacturer's instruction. After quantification using a Nanodrop spectrophotometer, 10 µg total RNA was reversed transcribed into cDNA using the Revert Aid First Strand cDNA Synthesis Kits (Fermentas), respectively. Besides, cDNA prepared respectively with Super Array PCR master mix (Cat. No. PA-112) was needed in order to perform PCR Array.2 Profiler PCR Array, including 84 key genes encoding glycan processing enzymes i.e. glycosyltransferase and glycosidase for several important sugars: galactose, glucose, mannose, N-acetylgalactosamine, N-acetylglucosamine, fucose and sialic acid, was designed and produced by the Qiagen company (German).The Human Glycosylation RTThe PCR Array and qRT-PCR were performed on IQ5 machine (Bio-Rad) and the PCR cycling condition was set as follows: 95°C for 5 min, 40 cycles of 95°C for 15 s, 60°C for 15 s and 72°C for 20 s. Each test was run three times and the mean was taken to eradicate any discrepancies.The used primer sequences in qRT-PCR were summarized in Take advantage of the Ct obtained from PCR-Array, the MeV 4.8.1 was used to map the hierarchical clustering of all the glycogenes in the PCR Array, the differentially transcribed glycogenes generated and altered N-linked glycans, O-linked glycans and glycosphingolipids, respectively.β-Actin, and was calculated respectively for each cell using 2−ΔΔCt, where ΔΔCt = (Ct target gene-β-Actin) MHCC97L/MHCC97H/HCCLM3-(Ct target gene-β-Actin) Hep3B. Glycogene with differences greater than 2-fold (P≤0.05) was considered as the differentially transcribed glycogenes and they were divided into six clusters by a K-means approach, which is a kind of clustering algorithm for grouping genes or proteins with a similar expression pattern β-Actin was as the reference and the relative expressions value were expressed by ΔCt = Ct target gene-β-Actin, which means that the higher value of ΔCt of the glycogene, the lower its transcription level.The fold change was normalized to the expression of the housekeeping gene, 2 Profiler PCR Array and was used to produce a hierarchical clustering scheme  compared with the non-metastatic HCC cell line Hep3B, while fifteen mRNA like c1galt1, gcent4, man1a1 were down-regulated  and their orthotopic xenograft tumors. The housekeeping gene β-Actin was as the reference and normalized the obtained fold changes of differential glycogenes. The results of the human HCC cell lines were shown in c1ganlt1, man1a1, mgat4a, galnt3 were all down-regulated whereas gcnt3, mgat5, mgat5b were all obviously up-regulated, almost all the transcription levels of the glycogenes had significant difference. These results supported the transcriptional profiling from the PCR Array.Seven glycogenes: K-means clustering  had more influence could be discriminated by the p-value of overlap. Like edem1 as a glycogene was influenced by XBP1, SIRT1, TMBIM6, IDH1, BAK1, KCNIP3, nevertheless, KCNIP3 and TMBIM6 play much more important roles. For the seventeen glycogenes in the regulatory network, NCF4, SDC1, SIRT1, CLOCK, TMBIM6 had smaller p-values and it suggested that they may play more important roles in the transcriptional regulation of the differentially transcribed glycogenes.All of the thirty-three differentially transcribed glycogenes were uploaded to the database to analyze upstream regulatory events and build the possible regulatory network by the IPA . The molgalnt3, gcnt3, man1a1, mgat5b were selected to detect the expressions in clinical cancer specimens and their results were shown in galnt3 and man1a1 in metastatic HCC clinical cancer specimens were lower than in the non-metastatic ones, there were no significant differences . While the divergent trends of gcnt3 and mgat5b between non-metastatic and metastatic HCC clinical cancer specimens were statistically significant  and they were all up-regulated in metastatic HCC clinical cancer specimens, which trends were as same as in human HCC cell lines and their orthotopic xenograft tumors. gcnt3 (glucosaminyl (N-acetyl) transferase 3, mucin type) is required to form GlcNAcβ1→6Gal/GalNAc -glycoprotein β-1,6-N-acetyl-glucosaminyltransferase, isozyme B) acts on the GlcNAc β1,2-Manα1-Ser/Thr moiety, forms a 2,6-branched structure in brain O-mannosyl glycan  as a criteria. A glycomics study using glycogene microarray which contained 115 genes by Kang et.al reported eighteen glycogenes were up-regulated in high metastatic potential HCCLM3 cell lines in comparison to Hep3B cell lines, while eleven glycogenes were down-regulated b3galt1, fut8, gal3st2, mgat5, st3gal5 and st6gal1 were overexpressed in MHCC97H cell lines compared with those in MHCC97L cell lines; conversely, five glycogenes, b3gnt6, b3gnt7, galnt6, mgat3 and st8sia2 were down-regulated mgat1, mgat5 and st3gal1, some glycogenes such as “UDP-N-acetyl-α-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase” family members: galnt3, galnt4, galnt11, galnt13, and “mannosidase, α class” family members, for instance, man1b1, man1c1, man2a2, their different expressions were associated with HCC metastasis are the first reported. This research revealed that glycogenes which could generate and alter sialylated glycan structures or β-1, 6-GlcNAc branched N-Glycan were significantly up-regulated in the metastatic HCC cell lines and their orthotopic xenograft tumors. Sialic acids could prevent cell-cell interactions through charge repulsion effects mgat5 could advance tumor growth and metastasis by destructing the extracellular matrix or boost the production of angiogenic factors by a metal ion-dependent serine protease. mgat5 could also promote tumor metastasis by catalyzing sugar chain which can change the function of angiogenic factors or by directly increasing the gene transcription of angiogenic factors The metastatic potential of HCC cell lines in our experiments were strengthening step by step. Hep3B, the non-metastatic cell line from ATCC, was established from a patient of 8-year-old black boy. Nevertheless, MHCC97L, MHCC97H and HCCLM3, all of them can metastasize, whose metastatic potential were increasing in general The qRT-PCR analysis of the transcribed glycogenes in the human HCC cell lines and their orthotopic xenograft tumors provided evidences of the same changing trends in statistics (P<0.05) to support the analysis of PCR-array results. Nevertheless, we found that the respective fold-change value of comparison results within metastatic HCC cell lines and their orthotopic xenograft tumors had no obvious gradually change trend. It revealed that the transcriptional of glycogenes was highly related with metastasis no matter whether their metastatic potentials were high or low. Therefore, we suggested that our future studies about glycosyltransferases should more focus on comparison of whether these cells or orthotopic xenograft tumors were able to metastatic or not rather than the difference of their metastatic potentials.fut8 levels and also to affect core fucosylation of secreted proteins galnt7, which regulates metabolism of liver toxin galactosamine The regulatory network of part of the differentially transcribed glycogenes was obtained by the IPA, and it included six types of relationship and nineteen upstream regulators. Aside from regulators, current data suggests that miRNA is related with the expressions of glycogenes. Human and rodents HCC cell lines were transfected with miR-122 and miR-34a and the result demonstrated that forced expression of these miRNAs was able to induce a decrease of gcnt3 and mgat5b were associated with HCC metastasis.The validation in clinical cancer specimens showed some differences of the transcription levels of glycogenes in clinical tissue samples were not significant. It might be caused by the part of the differences among cell lines, orthotopic xenograft tumors and clinical tissue samples themselves. But it was important to note there were still a substantial part of glycogenes which were differentially transcribed between non-metastatic and metastatic tissues. This evidence suggested there might be some potential novel drug targets and biological markers to predict metastasis of HCC in the differentially transcribed genes. To validate the transcription levels of the remaining differentially transcribed glycogenes, further investigation is needed and this part of the work is undergoing. Moreover, it could not be neglected that our study is the first time to provide evidences in human HCC cell lines, orthotopic xenograft tumors and clinical tissue samples that the up-regulated of 2 Profiler PCR Array. A total of seven glycogenes, including c1galt1, galnt3, gcnt3, man1a1, mgat4b, mgat5, mgat5b, were further identified by qRT-PCR in HCC cell lines and their orthotopic xenograft tumors. All of these differentially transcribed glycogenes were analyzed by K-means and GO analysis, and were uploaded to the IPA database for regulatory network analysis. What was more, galnt3, gcnt3, man1a1 and mgat5b were selected to further validation in clinical cancer specimens and this is the first report that the up-regulated of gcnt3 and mgat5b were associated with HCC metastasis. The differentially transcribed glycogenes could be the potential targets for the novel drugs and might act as the potential biological markers to predict metastasis of HCC.In summary, this study identified thirty-three differentially transcribed glycogenes by the Human Glycosylation RTTable S1General information of non-metastatic and metastatic HCC patients.(DOCX)Click here for additional data file.Table S2The K-means class of the differentially expression glycogenes.(DOCX)Click here for additional data file.


High numbers of tumour infiltrating lymphocytes has been associated with a significantly improved prognosis. Some of the key cell types involved are Cork Cancer Research Centre (CCRC) has long utilised an ablative cancer treatment that involves delivering a pulse of electrical energy (electroporation) directly to the tumour. This treatment results in the formation of reversible pores in the cell membrane, allowing up to a 1000 fold increase in chemotherapy drug absorption (Electrochemotherapy). A significant benefit of this non thermal ablation is the release of tumour associated antigens into the microenvironment, leading to activation of APCs and subsequent cross presentation of the antigens to circulating T and B cells. While ECT facilitates the priming of a favourable immune response, the combination of iCOS antibody, capable of stimulating T cell proliferation, causes a very robust mediated regression of treated tumours. In addition, ICOS is involved in humoral immune responses  and increases the production of IL-4.+ and CD8+ T cells, CD45R+ B cells and CD11c+ DCs, post treatment.Preclinical work in aggressive murine models has yielded very encouraging results. The combinational therapy has achieved cures in both colorectal (CT26) and metastatic lung (LLC) cancer cell lines. Follow up immunohistochemical staining confirmed the presence of tumour infiltrating CD3


Mechanisms involved in regulating metabolic reprogramming in cancer cells are not fully understood. Acetylation is emerging as a major regulator of mitochondrial metabolism and may contribute to metabolic derangements that occur in cancer cells. Sirtuin-3, (SIRT3), is the main mitochondrial deacetylase and it serves to maintain mitochondrial energy homeostasis by deacetylating and activating mitochondrial proteins. Loss of SIRT3 leads to altered cellular metabolism including reduced ATP production and decreased fatty acid oxidation . RemarkaStable cell lines expressing shScramble or shSIRT3 were made by lentiviral transduction in HepG2 and 293T cells. Metabolomic analysis of stable cell lines were done by GC/MS and MS/MS. Glutamine oxidation was measured by treating cells with 14C glutamine and capturing radiolabeled CO2.Protein acetylation is particularly sensitive to nutrient status, such as high fat diet feeding . Our datThese data illustrate an anabolic shift in cell metabolism that is required to supply biosynthetic precursors necessary for rapid cell growth. We believe that loss of SIRT3 and subsequent hyperacetylation of mitochondrial proteins leads to a rerouting of energetic substrates supporting oncogenically driven tumor growth.