Limitation of sulfur-containing amino acids perturbs the oxidoreductive balance, particularly in gene -encoding the methionine transporter SBAT1- and reduced methionine uptake. Results Methionine limitation reduces growth of Ras-transformed mouse fibroblasts more than growth of normal cells First, we analyzed cell proliferation of normal NIH3T3 and Ras-transformed NIH-RAS mouse fibroblasts under standard growth condition (0.2 mM Cys, 0.2 mM Met), limitation (1/8: 0.025 mM; 1/4: 0.05 mM; 1/2: 0.1 mM) and deprivation of cysteine or methionine. human is usually active essentially only in cells from splanchnic organs. Here we exhibited that mouse embryonic fibroblasts are not able to convert methionine into cysteine. For this reason the trans-sulfuration reaction is usually highlighted in grey.(PDF) pone.0163790.s001.pdf (235K) GUID:?9E2A8C7F-B317-47BC-B783-E6149454E7EC S2 Fig: Ras and MAPK activation state and expression levels in cellular models used in the paper: NIH3T3, NIH-RAS, NIH-RAS pGEF-DN and NIH-RAS pcDNA3. Expression levels of Total Ras proteins (A) and MAPKs p42 and p44 (B) in cell lysates of pull down assay. Antibodies directed against Ras (sc259 Santa Cruz), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling #9101) and p44/42 MAPK (Erk1/2) (Cell Signaling #9102) were used. (C) PT2977 RasCGTP eluted from GSTCRBDCglutathioneCsepharose, pre-incubated with cell lysates. Pull down assay was performed as explained in [7]. (D) Quantification of the RasCGTP amount after normalization over total Ras. Data are normalized over the Ras-GTP/total Ras ratio in NIH3T3 taken equal to 100. Data shown are imply +/- standard deviation of two impartial experiments. (E) Morphological analysis of the different cell lines. (F) Phospho-p44/42 MAPK level in cell lysates, determined by ELISA assay performed using PathScan? Phospho-p44/42 MAPK (Thr202/Tyr204) (Cell Signaling). Data shown are imply +/- standard deviation of two impartial experiments. (F) 100X magnification of a generated by NIH-RAS cells in formation assay shown in Fig 1.(PDF) pone.0163790.s002.pdf (395K) GUID:?72F65A17-F168-4921-ADDB-62566C1E3FC9 S3 Fig: Over-expression of GEF-DN reverts sensitivity to methionine limitation in NIH-RAS cells and partially rescues the defect in the expression of gene encoding methionine transporter SBAT1. (A) Cell proliferation of NIH3T3, NIH-RAS, NIH-RAS pGEF-DN and NIH-RAS pcDNA3 cells produced in media with different concentrations of methionine and counted daily for 72 h of growth under conditions indicated. Plotted data are mean +/- standard deviation. computed from three impartial experiments. (B) Relative to t = 0 cell proliferation of NIH3T3, NIH-RAS, NIH-RAS pGEF-DN and NIH-RAS pcDNA3 cells produced for 72 h in media with different PT2977 concentrations of methionine, as indicated in (A). Part of the data in (B) are present in Fig 1D. (C) Semi-quantitative RT-PCR Rabbit polyclonal to KLK7 results for NIH3T3, NIH-RAS, NIH-RAS pGEF-DN and NIH-RAS pcDNA3 cells produced for 48 h in standard medium performed in triplicate on genes showing at least a two-fold switch between NIH-RAS normal cells (here represented in strong), a two-fold and a 0.05 cut-offs on Fold Changes and on oncogene activation in NIH3T3 mouse fibroblasts on transfer and metabolism of cysteine and methionine. We show that cysteine limitation and deprivation cause apoptotic cell death (cytotoxic effect) in both normal and geneencoding the nutrient transporter SBAT1, known to exhibit a strong preference for methionineand decreased methionine uptake. Conclusions and Significance Overall, limitation of sulfur-containing amino acids results in a more dramatic perturbation of the oxido-reductive balance in proto-oncogene [1,2,3,4] has a great incidence in human tumors, as reported in the catalogue of somatic mutations in malignancy (COSMIC) [5]. activation occurs in 22% of all tumors, prevalently PT2977 in pancreatic carcinomas (about 90%), colorectal carcinomas (40C50%), and lung carcinomas (30C50%), as well as in biliary tract malignancies, endometrial malignancy, cervical malignancy, bladder cancer, liver malignancy, myeloid leukemia and breast malignancy. K-Ras oncoproteins are important clinical targets for anti-cancer therapy [6] and several strategies have been explored in order to inhibit aberrant Ras signaling, as examined in [7,8,9,10]. The acquisition of important hallmark characteristics of malignancy cells, including enhanced cell growth and survival, rely on deep changes in metabolism driven by oncogene activation [11,12,13,14,15]. Oncogenic activation of contributes to the acquisition of the hyper-glycolytic phenotype (also known as Warburg effect, from your pioneering studies of Warburg [16]) due to enhancement in glucose transport and aerobic glycolysis [17,18]. oncogene activation also correlates with down-regulated expression of mitochondrial genes, altered mitochondrial morphology and production of large amount of reactive oxygen species (ROS) associated with mitochondrial metabolism [19,20]. Furthermore, activation allows cells to make extensive anaplerotic usage of glutamine, the more concentrated amino acid in human plasma [21]. In Ras-transformed cells, glutamine is largely utilized through reductive carboxylation that results in a non-canonical tricarboxylic acid cycle (TCA) pathway [19,22,23,24,25,26]. These metabolic changes render Ras-transformed cells addicted to glutamine, and to glutaminolysis, and offer new therapeutic opportunities. Indeed, glutamine metabolism restriction and targeted malignancy therapeutics directed against glutamine transporters or glutaminolysis can be used to limit tumor cell proliferation and survival without affecting normal cells [27,28,29]. Besides glutamine transporters, all amino acid transporters are being receiving attention from scientific community as potential drug targets for malignancy treatment, given the increased demand of malignancy cells for these nutrients to support their enhanced cell growth [30,31]..