The Experts below are selected from a list of 168 Experts worldwide ranked by ideXlab platform
Richard G Moran - One of the best experts on this subject based on the ideXlab platform.
-
A Defect in the p53 Response Pathway Induced by de Novo Purine Synthesis Inhibition
The Journal of biological chemistry, 2003Co-Authors: Julie L. Bronder, Richard G MoranAbstract:Abstract p53 is believed to sense cellular ribonucleotide depletion in the absence of DNA strand breaks and to respond by imposition of a p21-dependent G1 cell cycle arrest. We now report that the p53-dependent G1 checkpoint is blocked in human carcinoma cell lines after inhibition of de novo Purine Synthesis by folate analogs inhibitory to glycinamide ribonucleotide formyltransferase (GART). p53 accumulated in HCT116, MCF7, or A549 carcinoma cells upon GART inhibition, but, surprisingly, transcription of several p53 targets, including p21cip1/waf1, was impaired. The mechanism of this defect was examined. The p53 accumulating in these cells was nuclear but was not phosphorylated at serines 6, 15, and 20, nor was it acetylated at lysines 373 or 382. The DDATHF-stabilized p53 bound to the p21 promoter in vitro and in vivo but did not activate histone acetylation over the p53 binding sites in the p21 promoter that is an integral part of the transcriptional response mediated by the DNA damage pathway. We concluded that the robust initial response of the p53 pathway to GART inhibitors is not transcriptionally propagated to target genes due to a defect in p53 post-translational modifications and a failure to open chromatin structure despite promoter binding of this unmodified p53.
-
Antifolates targeting Purine Synthesis allow entry of tumor cells into S phase regardless of p53 function.
Cancer research, 2002Co-Authors: Julie L. Bronder, Richard G MoranAbstract:The class of folate antimetabolites typified by (6R)-dideazatetrahydrofolate (lometrexol, DDATHF) are specific inhibitors of de novo Purine Synthesis because of potent inhibition of glycinamide ribonucleotide formyltransferase (GART) but do not induce detectable levels of DNA strand breaks. As such, they are a test case of the concept that ribonucleotide depletion can be sensed by p53, resulting in a G1 cell cycle block. The GART inhibitors have been proposed previously to be cytotoxic in tumor cells lacking p53 function but only cytostatic in p53 wild-type tumor cells. We have investigated this concept. Cell cycle progression into and through S phase was slowed by DDATHF, but both p53 +/+ and −/− human colon carcinoma cells entered and completed one S phase in the presence of drug. This inability of p53 to initiate a G1 arrest after DDATHF treatment was mirrored by an independence of the cytotoxicity of DDATHF on p53 function. We conclude that carcinoma cells are killed equally well by DDATHF and related compounds whether or not the p53 pathway is intact and that the utility of GART inhibitors would not be limited to p53-negative tumors.
-
Analysis of a Mouse Gene Encoding Three Steps of Purine Synthesis Reveals Use of an Intronic Polyadenylation Signal without Alternative Exon Usage
The Journal of biological chemistry, 1995Co-Authors: Julie L. C. Kan, Richard G MoranAbstract:Abstract A single mouse genomic locus encodes proteins catalyzing three steps of Purine Synthesis, glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS), and glycinamide ribonucleotide formyltransferase (GART). This gene has 22 exons and spans 28 kilobases. The existence of a second genetic locus and closely related pseudogenes was ruled out by Southern analysis. Mouse tissues express two related classes of messages encoded by this single locus: a trifunctional GARS-AIRS-GART mRNA and a monofunctional GARS mRNA. These transcripts used the same set of multiple transcriptional start sites, and both used the same first 10 exons. CCAAT and TATA elements were not found for this locus. Exon 11, which represented the last coding sequence of the GARS domain, was differentially utilized for the two messages. The trifunctional mRNA was generated by splicing exon 11 to exon 12, the first coding sequence for the AIRS domain with subsequent use of a polyadenylation signal at the end of exon 22. Genomic sequence corresponding to the 3′-UTR of the monofunctional GARS mRNA was contiguous with exon 11, so that the smaller message arose from the recognition of one of the multiple polyadenylation signals present within the intron between exons 11 and 12. Hence, polyadenylation of the primary transcript at a position corresponding to an intron of the genomic locus was responsible for the generation of the monofunctional GARS class of mRNAs. This utilization of an intronic polyadenylation site without alternative exon usage is comparable to the mechanism whereby both secreted and membrane-bound forms of the immunoglobulin μ heavy chain are made from a single genetic locus.
-
Mouse cDNAs encoding a trifunctional protein of de novo Purine Synthesis and a related single-domain glycinamide ribonucleotide synthetase.
Gene, 1993Co-Authors: Julie L. C. Kan, Mehrdad Jannatipour, Shirley M. Taylor, Richard G MoranAbstract:Three of the enzymatic activities of de novo Purine Synthesis, glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS) and glycinamide ribonucleotide formyltransferase (GART), can be catalyzed by a single 110-kDa protein in mouse cells. Western blots using a polyclonal antibody (Ab) to this protein identified two species, the trifunctional 110-kDa protein and a 50-kDa cytosolic protein with GARS, but not GART activity. We used Ab and, subsequently, oligodeoxyribonucleotide screens to isolate cDNAs corresponding to these two proteins from mouse T-cell cDNA expression libraries. The sequence of one class of these cDNAs and the partial sequence of a corresponding genomic clone defined an open reading frame (ORF) encoding a 1010-amino-acid (aa) protein, individual domains of which showed high homology to each of the monofunctional bacterial GARS, AIRS and GART proteins, and to each domain of chicken and human trifunctional GARS-AIRS-GARTs. cDNAs corresponding to the smaller protein contained a 1.3-kb ORF with complete identity to the GARS domain of, but with a 3' untranslated region different from, the trifunctional cDNAs. Hence, both cDNAs appear to derive from the same gene due to either differential splicing or use of an intronic polyadenylation signal. The functional requirement for the expression of both trifunctional protein with GARS activity and monofunctional, catalytically active GARS is unknown.
-
Cytotoxicity of antifolate inhibitors of thymidylate and Purine Synthesis to WiDr colonic carcinoma cells.
Cancer research, 1993Co-Authors: Scott G. Smith, Norman L. Lehman, Richard G MoranAbstract:We have studied the cytotoxicity of 5,10-dideazatetrahydrofolate (DDATHF) and of D-1694 to human WiDr colonic carcinoma cells as a model system for the effects of pure inhibitors of either the de novo Purine Synthesis pathway or thymidylate Synthesis. The growth of this cell line was inhibited by very low concentrations of either agent and the lethality of DDATHF and D-1694 was completely prevented by continuous exposure to either hypoxanthine or thymidine, respectively, indicating that these compounds were very potent metabolic inhibitors, each specific for one of these pathways. D-1694 was highly cytotoxic (> 3 logs of kill) after a 4-h exposure to 1 microM drug, or a 24-h exposure to very low concentrations (0.04 microM). On the other hand, the cytotoxicity of DDATHF was substantially lower, with 2 logs of cell kill requiring >> 100 microM with 4 h of exposure or approximately 40 microM for 72 h of exposure. Maximal cell kill induced by D-1694 was 5-6 logs, consistent with elimination of all viable cells except preexisting mutants. A maximum of 2-3 logs of cell kill was observed with DDATHF. Exposure of WiDr cells to either D-1694 or DDATHF caused striking cellular changes, but the morphologies of cells treated with the two drugs were remarkably different. D-1694-treated cells detached from the dish within 1-2 days after a megaloblastosis, whereas DDATHF-treated cells remained adherent to the dishes for at least 10 days after treatment. The addition of thymidine to D-1694-treated cultures or hypoxanthine to DDATHF-treated cells after up to 20 h of drug exposure completely prevented cytotoxicity of either drug. With longer exposures, cytotoxicity of both drugs progressively increased in spite of such rescue. Our results indicate that substantial (99-99.9%) tumor cell kill can be induced by a pure inhibitor of Purine Synthesis, but that the rate of commitment to cell death and the extent of cell kill is greater with a pure inhibitor of thymidylate Synthesis.
Oded Sperling - One of the best experts on this subject based on the ideXlab platform.
-
Cyclic AMP decreases the availability of 5-phosphortoosyl-1-pyrophosphate and decelerates de novo Purine Synthesis in rat hepatocytes
Life sciences, 1998Co-Authors: Pnina Boer, Shamai Giler, Oded SperlingAbstract:Abstract Cyclic adenosine monophosphate (cAMP) was found to decrease the availability of 5-phosphoribosyl-1-pyrophosphate (PRPP) and to decelerate the rate of de novo Purine Synthesis in suspensions of adult rat hepatocytes. Glucagon did not affect these parameters. The glucagon antagonist des-His 1 [Glu 9 ]glucagon amide (DHGA), and the protein kinase C activator 1,2-dioctanoyl-sn-glycerol (DOG) were also found to lower PRPP availability. Incubation of the hepatocytes with dbcAMP or with DHGA, did not alter the activity of PRPP synthetase in the hepatocyte lysates, indicating that the above effects are not mediated through the activity of this enzyme. The possibility that the decrease in PRPP availability reflects increased consumption associated with accelerated pyrimidine Synthesis is discussed. The decelerated rate of de novo Purine Synthesis is probably secondary to the decreased PRPP availability.
-
Acceleration of Purine Synthesis in mouse liver by glycogenolytic hormones.
Biochemical medicine and metabolic biology, 1991Co-Authors: Pnina Boer, Rivka Mamet, Oded SperlingAbstract:Administration (ip) into fed mice of glucagon, epinephrine, vasopressin, oxytocin, angiotensin II, and dibutyryl cyclic AMP (dbcAMP) resulted in a rapid (within 2.5 to 15 min) elevation of PRPP content (two- to threefold) and in acceleration of the rate of de novo Purine Synthesis (twofold). Inhibition of the epinephrine-stimulated glycogenolysis by 2,5-anhydromannitol diminished markedly the acceleration effect of the hormone on the rate of Purine Synthesis. Administration of the hormones caused a rapid rise in the liver content of glucose 6-phosphate (G6P) by 15-70% but did not increase the ribose 5-phosphate (R5P) content. Liver ATP content was not affected. The hormones did not cause direct activation of PRPP synthetase, as gauged by the specific activity of the enzyme, its Km for substrates R5P and ATP, and its sensitivity to inhibition by ADP and GDP. The hormones did not increase the liver content of the enzyme activators Pi and Mg2+. The results suggest that the glycogenolytic hormones accelerate Purine Synthesis by a metabolic mechanism associated with the enhancement of glycogenolysis. PRPP Synthesis is probably enhanced by the glycogenolysis-induced alterations in the cellular content of some metabolites other than R5P.
Gerry R Boss - One of the best experts on this subject based on the ideXlab platform.
-
Akt phosphorylation and regulation of transketolase is a nodal point for amino acid control of Purine Synthesis.
Molecular cell, 2014Co-Authors: Arindam Saha, Renate B. Pilz, Stephen Connelly, Jingjing Jiang, Shunhui Zhuang, Deron T. Amador, Tony Phan, Gerry R BossAbstract:The phosphatidylinositol 3-kinase (PI3K)/Akt pathway integrates environmental clues to regulate cell growth and survival. We showed previously that depriving cells of a single essential amino acid rapidly and reversibly arrests Purine Synthesis. Here we demonstrate that amino acids via mammalian target of rapamycin 2 and IκB kinase regulate Akt activity and Akt association and phosphorylation of transketolase (TKT), a key enzyme of the nonoxidative pentose phosphate pathway (PPP). Akt phosphorylates TKT on Thr382, markedly enhancing enzyme activity and increasing carbon flow through the nonoxidative PPP, thereby increasing Purine Synthesis. Mice fed a lysine-deficient diet for 2 days show decreased Akt activity, TKT activity, and Purine Synthesis in multiple organs. These results provide a mechanism whereby Akt coordinates amino acid availability with glucose utilization, Purine Synthesis, and RNA and DNA Synthesis.
-
Cell cycle regulation of Purine Synthesis by phosphoribosyl pyrophosphate and inorganic phosphate.
The Biochemical journal, 2013Co-Authors: Alla Fridman, Renate B. Pilz, Arindam Saha, Adriano Chan, Darren E. Casteel, Gerry R BossAbstract:Cells must increase Synthesis of Purine nucleotides/deoxynucleotides before or during S-phase. We found that rates of Purine Synthesis via the de novo and salvage pathways increased 5.0- and 3.3-fold respectively, as cells progressed from mid-G1-phase to early S-phase. The increased Purine Synthesis could be attributed to a 3.2-fold increase in intracellular PRPP (5-phosphoribosyl-α-1-pyrophosphate), a rate-limiting substrate for de novo and salvage Purine Synthesis. PRPP can be produced by the oxidative and non-oxidative pentose phosphate pathways, and we found a 3.1-fold increase in flow through the non-oxidative pathway, with no change in oxidative pathway activity. Non-oxidative pentose phosphate pathway enzymes showed no change in activity, but PRPP synthetase is regulated by phosphate, and we found that phosphate uptake and total intracellular phosphate concentration increased significantly between mid-G1-phase and early S-phase. Over the same time period, PRPP synthetase activity increased 2.5-fold when assayed in the absence of added phosphate, making enzyme activity dependent on cellular phosphate at the time of extraction. We conclude that Purine Synthesis increases as cells progress from G1- to S-phase, and that the increase is from heightened PRPP synthetase activity due to increased intracellular phosphate.
O Sperling - One of the best experts on this subject based on the ideXlab platform.
-
role of cellular ribose 5 phosphate content in the regulation of 5 phosphoribosyl 1 pyrophosphate and de novo Purine Synthesis in a human hepatoma cell line
Metabolism-clinical and Experimental, 1995Co-Authors: P. De Boer, O SperlingAbstract:Abstract 5-Phosphoribosyl-1-pyrophosphate (PRPP) is an important regulator of de novo Purine Synthesis. However, the role of ribose-5-phosphate (R5P), the precursor for PRPP, in the regulation of PRPP and de novo Purine Synthesis has not yet been clarified conclusively. This study was designed to clarify interrelationships between R5P content, PRPP availability, and the rate of de novo Purine Synthesis in the cultured human hepatoma cell line (HepG 2 ), a plausible model for normal human hepatocytes. Increasing glucose concentration in the culture media from 0 to 10 mmol/L resulted in a 2.9-fold elevation of cellular R5P content (from 107 ± 31 to 311 ± 57 nmol/g protein), associated with a correlated increase of 7.14-fold in cellular PRPP availability (from 4.76 ± 3.4 to 34 ± 8.4 pmol/mg protein/min) and of 149-fold in the rate of de novo Purine Synthesis (from 55 to 8,204 dpm/mg protein/h). Plotting the rate of de novo Purine Synthesis versus R5P content indicates that at a wide range of R5P content, including that prevailing in hepatocytes under physiological conditions, the rate of Purine Synthesis depends on R5P content. A similar dependence was also demonstrated for PRPP availability. The rate of de novo Purine Synthesis exhibited a sigmoidal dependence on PRPP availability. The demonstration in human hepatocytes of dependence of the rate of Purine Synthesis on R5P content has implications concerning the pathogenesis of Purine overproduction associated with several inborn and acquired conditions in man.
Dean R. Appling - One of the best experts on this subject based on the ideXlab platform.
-
Role of mitochondrial and cytoplasmic serine hydroxymethyltransferase isozymes in de Novo Purine Synthesis in Saccharomyces cerevisiae
Biochemistry, 1997Co-Authors: Evdokia Kastanos, Yakov Y. Woldman, Dean R. ApplingAbstract:One-carbon units are essential to a variety of anabolic processes which yield necessary cellular components including Purines, pyrimidines, amino acids, and lipids. Serine hydroxymethyltransferase (SHMT) is the major provider of one-carbon units in the cell. The other product of this reaction is glycine. Both of these metabolites are required in de novo Purine bioSynthesis. In Saccharomyces cerevisiae, mitochondrial and cytoplasmic SHMT isozymes are encoded by distinct nuclear genes (SHM1 and SHM2). Molecular genetic analyses have begun to define the roles of these two isozymes in folate-mediated one-carbon metabolism [McNeil, J. B., et al. (1996) Genetics 142, 371-381]. In our study, the SHM1 and SHM2 genes were disrupted singly and in combination to investigate the contributions of the two SHMT isozymes to the production of glycine and one-carbon units required in Purine bioSynthesis. Cell subfractionation experiments indicated that while only 5% of total activity was localized in the mitochondria, the specific activity in that compartment was much higher than in the cytoplasm. Growth and 13C NMR experiments indicate that the two isozymes function in different directions, depending on the nutritional conditions of the cell. When yeast was grown on serine as the primary one-carbon source, the cytoplasmic isozyme was the main provider of glycine and one-carbon groups for Purine Synthesis. When grown on glycine, the mitochondrial SHMT was the predominant isozyme catalyzing the Synthesis of serine from glycine and one-carbon units. However, when both serine and glycine were present, the mitochondrial SHMT made a significant contribution of one-carbon units, but not glycine, for Purine Synthesis. Finally, NMR data are presented that suggest the existence of at least two sites of de novo Purine bioSynthesis in growing yeast cells, each being fed by distinct pools of precursors.
