Vacuolar and cytoskeletal dynamics during elicitor-induced programmed cell death in tobacco BY-2 cells

Responses of plant cells to environmental stresses often involve morphological changes, differentiation and redistribution of various organelles and cytoskeletal network. Tobacco BY-2 cells provide excellent model system for in vivo imaging of these intracellular events. Treatment of the cell cycle-synchronized BY-2 cells with a proteinaceous oomycete elicitor, cryptogein, induces highly synchronous programmed cell death (PCD) and provide a model system to characterize vacuolar and cytoskeletal dynamics during the PCD. Sequential observation revealed dynamic reorganization of the vacuole and actin microfilaments during the execution of the PCD. We further characterized the effects cryptogein on mitotic microtubule organization in cell cycle-synchronized cells. Cryptogein treatment at S phase inhibited formation of the preprophase band, a cortical microtubule band that predicts the cell division site. Cortical microtubules kept their random orientation till their disruption that gradually occurred during the execution of the PCD twelve hours after the cryptogein treatment. Possible molecular mechanisms and physiological roles of the dynamic behavior of the organelles and cytoskeletal network in the pathogenic signal-induced PCD are discussed.Key words: actin microfilament, cell cycle, cryptogein, microtubules, nuclei, programmed cell death, tobacco BY-2 cells, vacuoles     

Regulation of purine synthesis de novo in human fibroblasts by purine nucleotides and phosphoribosylpyrophosphate

Previous studies of purine nucleotide synthesis de novo have suggested that major regulation of the rate of the pathway is affected at either the phosphoribosylpyrophosphate (PP-Rib-P) synthetase reaction or the amidophosphoribosyltransferase (amido PRT) reaction, or both. We studied control of purine synthesis de novo in cultured normal, hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient, and PP-Rib-P synthetase-superactive human fibroblasts by measuring concentrations and rates of synthesis of PP-Rib-P and purine nucleotide end products, proposed effectors of regulation, during inhibition of the pathway. Incubation of cells for 90 min with 0.1 mM azaserine, a glutamine antagonist which specifically blocked the pathway at the level of conversion of formylglycinamide ribotide, resulted in a 5-16% decrease in purine nucleoside triphosphate concentrations but no consistent alteration in generation of PP-Rib-P. During this treatment, however, rates of the early steps of the pathway were increased slightly (9-15%) in normal and HGPRT-deficient strains, more markedly (32-60%) in cells with catalytically superactive PP-Rib-P synthetases, and not at all in fibroblasts with purine nucleotide feedback-resistant PP-Rib-P synthetases. In contrast, glutamine deprivation, which inhibited the pathway at the amido PRT reaction, resulted in time-dependent nucleoside triphosphate pool depletion (26-43% decrease at 24 h) accompanied by increased rates of PP-Rib-P generation and, upon readdition of glutamine, substantial increments in rates of purine synthesis de novo. Enhanced PP-Rib-P generation during glutamine deprivation was greatest in cells with regulatory defects in PP-Rib-P synthetase (2-fold), but purine synthesis in these cells was stimulated only 1.4-fold control rates by glutamine readdition. Stimulation of these processes in normal and HGPRT-deficient cells and in cells with PP-Rib-P synthetase catalytic defects was, respectively: 1.5 and 2.0-fold; 1.5 and 1.7-fold; and 1.6 and 4.1-fold. These studies support the following concepts. 1) Rates of purine synthesis de novo are regulated at both the PP-Rib-P synthetase and amido PRT reactions by end products, with the latter reaction more sensitive to small changes in purine nucleotide inhibitor concentrations. 2) PP-Rib-P exerts its role as a major regulator of purine synthetic rate by virtue of its interaction with nucleotide inhibitors to determine the activity of amido PRT. 3) Activation of amido PRT by PP-Rib-P is nearly maximal at base line in fibroblasts with regulatory defects in PP-Rib-P synthetase.     

Nitric oxide modulates cadmium influx during cadmium-induced programmed cell death in tobacco BY-2 cells

Nitric oxide (NO) is a bioactive gas and functions as a signaling molecule in plants exposed to diverse biotic and abiotic stresses including cadmium (Cd²⁺). Cd²⁺ is a non-essential and toxic heavy metal, which has been reported to induce programmed cell death (PCD) in plants. Here, we investigated the role of NO in Cd²⁺-induced PCD in tobacco BY-2 cells (Nicotiana tabacum L. cv. Bright Yellow 2). In this work, BY-2 cells exposed to 150 μM CdCl₂ underwent PCD with TUNEL-positive nuclei, significant chromatin condensation and the increasing expression of a PCD-related gene Hsr203J. Accompanied with the occurring of PCD, the production of NO increased significantly. The supplement of NO by sodium nitroprusside (SNP) had accelerated the PCD, whereas the NO synthase inhibitor Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME) and NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) alleviated this toxicity. To investigate the mechanism by which NO exerted its function, Cd²⁺ concentration was measured subsequently. SNP led more Cd²⁺ content than Cd²⁺ treatment alone. By contrast, the prevention of NO by l-NAME decreased Cd²⁺ accumulation. Using the scanning ion-selective electrode technique, we analyzed the pattern and rate of Cd²⁺ fluxes. This analysis revealed the promotion of Cd²⁺ influxes into cells by application of SNP, while l-NAME and cPTIO reduced the rate of Cd²⁺ uptake or even resulted in net Cd²⁺ efflux. Based on these founding, we concluded that NO played a positive role in CdCl₂-induced PCD by modulating Cd²⁺ uptake and thus promoting Cd²⁺ accumulation in BY-2 cells.     

Characterization of the programmed cell death induced by metabolic products of Alternaria alternata in tobacco BY-2 cells

Alternaria alternata has received considerable attention in current literature and most of the studies are focused on its pathogenic effects on plant chloroplasts, but little is known about the characteristics of programmed cell death (PCD) induced by metabolic products (MP) of A. alternata, the effects of the MP on mitochondrial respiration and its relation to PCD. The purpose of this study was to explore the mechanism of MP-induced PCD in non-green tobacco BY-2 cells and to explore the role of mitochondrial inhibitory processes in the PCD of tobacco BY-2 cells. MP treatment led to significant cell death that was proven to be PCD by the concurrent cytoplasm shrinkage, chromatin condensation and DNA laddering observed in the cells. Moreover, MP treatment resulted in the overproduction of reactive oxygen species (ROS), rapid ATP depletion and a respiratory decline in the tobacco BY-2 cells. It was concluded that the direct inhibition of the mitochondrial electron transport chain (ETC), alternative pathway (AOX) capacity and catalase (CAT) activity by the MP might be the main contributors to the MP-induced ROS burst observed in tobacco BY-2 cells. The addition of adenosine together with the MP significantly inhibited ATP depletion without preventing PCD; however, when the cells were treated with the MP plus CAT, ROS overproduction was blocked and PCD did not occur. The data presented here demonstrate that the ROS burst played an important role in MP-induced PCD in the tobacco BY-2 cells.     

Hydrogen peroxide induces programmed cell death features in cultured tobacco BY-2 cells, in a dose-dependent manner

Active oxygen species (AOS), especially hydrogen peroxide, play a critical role in the defence of plants against invading pathogens and in the hypersensitive response (HR). This is characterized by the induction of a massive production of AOS and the rapid appearance of necrotic lesions is considered as a programmed cell death (PCD) process during which a limited number of cells die at the site of infection. This work was aimed at investigating the mode of cell death observed in cultures of BY-2 tobacco cells exposed to H(2)O(2). It was shown that H(2)O(2) is able to induce various morphological cell death features in cultured tobacco BY-2 cells. The hallmarks of cell death observed with fluorescent and electron microscopy differed greatly with the amount of H(2)O(2) added to the cell culture. The appearance of nuclear fragmentation similar to 'apoptotic bodies' associated with a fragmentation of the nuclear DNA into small fragments appear for almost 18% of the cells treated with 12.5 mM H(2)O(2). The early stages of the induction of this PCD process consisted in cell shrinkage and chromatin condensation at the periphery of the nucleus. Above 50 mM, H(2)O(2) induces high necrotic cell death. These data suggest that H(2)O(2)-induced cell damage is associated with the induction of various cell death processes that could be involved differently in plant defence reactions.     

Lipid-binding form is a key conformation to induce a programmed cell death initiated in tobacco BY-2 cells by a proteinaceous elicitor of cryptogein

Cryptogein, a proteinaceous elicitor secreted by Phytophthora cryptogea , induces a remarkable hypersensitive cell death in tobacco cells. Two cryptogein mutants were analysed to characterize the induction mechanism of cell death; one was a newly synthesized mutant N93A whose 93rd Asn residue was changed to Ala, the other was K13V whose Lys at position 13 was replaced with Val. The effect of these mutations was evaluated in terms of extracellular alkalization, production of active oxygen species (AOS) and progression to death. The mutation N93A resulted in a reduction in activity to 71.0, 74.6 and 24.5% for original rates of extracellular alkalization, AOS production and cell death progression, respectively. In the case of the K13V mutation, these rates changed to 114, 3.38 and 7.40%, respectively. The lipid-binding activities of the mutants were analysed using fluorogenic lipid of dehydroergosterol. The results for N93A and K13V were 38.3 and 3.40% compared with the wild type, respectively. These findings indicate that the lipid-binding form was the only conformation to induce the production of AOS and programmed cell death in plants.     

Nitric oxide production is not required for dihydrosphingosine-induced cell death in tobacco BY-2 cells

Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid Long Chain Base (LCB) in plants, is known to induce a calcium-dependent programmed cell death (PCD) in tobacco BY-2 cells. We have recently shown that DHS triggers a production of H₂O₂, via the activation of NADPH oxidase(s). However, this production of H₂O₂ is not correlated with the DHS-induced cell death but would rather be associated with basal cell defense mechanisms. In the present study, we extend our current knowledge of the DHS signaling pathway, by demonstrating that DHS also promotes a production of nitric oxide (NO) in tobacco BY-2 cells. As for H₂O₂, this NO production is not necessary for cell death induction.Key words: tobacco BY-2 cells, sphingolipids, LCBs, dihydrosphingosine, sphinganine, apoptosis, programmed cell death (PCD), nitric oxide (NO)These last few years, it has been demonstrated in plants that long chain bases (LCBs), the sphingolipid precursors, are important regulators of different cellular processes including programmed cell death (PCD).^1–3 Indeed, plant treatment with fumonisin B1 or AAL toxin, two mycotoxins that disrupt sphingolipid metabolism, leads to an accumulation of the dihydrosphingosine (d18:0, DHS), one of the most abundant free LCB in plants and correlatively to the induction of cell death symptoms.^4,5 A more recent study shows a rapid and sustained increase of phytosphingosine (t18:0), due to a de novo synthesis from DHS, when Arabidopsis thaliana leaves are inoculated with the avirulent strain Pseudomonas syringae pv. tomato (avrRpm1), known to induce a localized PCD called hypersensitive response (HR).⁶ More direct evidences were obtained from experiments on Arabidopsis cells where external application of 100 µM C2-ceramide, a non-natural acylated LCB, induced PCD in a calcium (Ca²⁺)-dependent manner.⁷ Recently, we have shown that DHS elicited rapid Ca²⁺ increases both in the cytosol and the nucleus of tobacco BY-2 cells and correlatively induced apoptotic-like response. Interestingly, blocking nuclear Ca²⁺ changes without affecting the cytosolic Ca²⁺ increases prevented DHS-induced PCD.⁸Besides calcium ions, reactive oxygen species (ROS) have also been suggested to play an important role in the control of PCD induced by sphingolipids in plants.⁹ Thus, the C2-ceramide-induced PCD in Arabidopsis is preceded by an increase in H₂O₂.⁷ However, inhibition of ROS production by catalase, a ROS-scavenging enzyme, did not prevent C2-ceramide-induced cell death, suggesting that this PCD is independent of ROS generation. Moreover, we recently showed in tobacco BY-2 cells that DHS triggers a dose-dependent production of H₂O₂ via activation of a NADPH oxidase.¹⁰ The DHS-induced cytosolic Ca²⁺ transient is required for this H₂O₂ production while the nuclear calcium variation is not necessary. In agreement with the results of Townley et al. blocking the ROS production using diphenyleniodonium (DPI), a known inhibitor of NADPH oxidases, does not prevent DHS-induced cell death. Gene expression analysis of defense-related genes, using real-time quantitative PCR (RT-qPCR) experiments, rather indicates that H₂O₂ generation is likely associated with basal defense mechanisms.¹⁰In the present study, we further investigated the DHS signaling cascade leading to cell death in tobacco BY-2 cells, by evaluating the involvement of another key signaling molecule i.e., nitric oxide (NO). In plants, NO is known to play important roles in numerous physiological processes including germination, root growth, stomatal closing and adapative response to biotic and abiotic stresses (reviewed in ref. ^11–14). NO has also been shown to be implicated in the induction of PCD in animal cells,¹⁵ in yeast,¹⁶ as well as in plant cells, in which it is required for tracheid differentiation¹⁷ or HR activation.^18,19 Interestingly in the latter case, the balance between NO and H₂O₂ production appears to be crucial to induce cell death.²⁰ Here we show in tobacco BY-2 cells that although DHS elicits a production of NO, this production is not necessary for the induction of PCD.     

Protein changes during programmed cell death in tobacco

Programmed cell death (PCD) was induced by the Yariv reagent in Nicotiana tabacum cv. Bright Yellow-2 cell suspension. The analyses of proteins extracts by 2-D electrophoresis clearly show massive protein degradation which was mainly due to cysteine protease activity. In contrast, some proteins remained unchanged up to 72 h after PCD induction. Peptide mass fingerprints of these proteins, obtained by MALDI-TOF, identified calreticulin, heat shock protein (HSP) 60, HSP70, malate dehydrogenase and mitochondrial ATP synthase β-subunit.     

Cytokinin-induced cell death is associated with elevated expression of alternative oxidase in tobacco BY-2 cells

N⁶-benzyladenine (BA) and N⁶-benzyladenosine ([9R]BA) induce massive production of reactive oxygen species (ROS) that is eventually followed by a loss of cell viability in tobacco BY-2 cells (Mlejnek et al. Plant Cell Environ 26:1723–1735, 2003, Plant Sci 168:389–395, 2005). Results presented in this work suggest that the main sources of ROS are likely mitochondria and that the maintenance of the mitochondrial transmembrane potential is crucial for ROS production in cytokinin-treaded BY-2 cells. Therefore, the possible involvement of alternative oxidase (AOX) in cell death process induced by BA and [9R]BA was studied. About three- to fourfold increase in mRNA levels of AOX1 was observed a few hours after the BA and [9R]BA addition into the growth medium. The elevated expression of AOX1 mRNA could be prevented by adding adenine and adenosine which simultaneously reduced the cytotoxic effects of BA and [9R]BA, respectively. N⁶-benzyladenine 7-β-d-glucoside ([7G]BA) which is a common non-toxic metabolite of BA and [9R]BA did not affect the AOX1 mRNA expression. Although AOX1 seemed to be involved in protection of BY-2 cells against the abiotic stress induced by BA and [9R]BA, the results do not support the idea that it protects cells from death exclusively by scavenging of reactive oxygen species. Indeed, N-propyl gallate, an inhibitor of AOX, decreased cell survival despite it concomitantly decreased the ROS production. This finding is in contrast to the effect of salicylhydroxamic acid, another well-known inhibitor of AOX, which also increased the number of dying cells while it increased the ROS production.     

Crosstalk between elicitor-induced cell death and cell cycle regulation in tobacco BY-2 cells

The molecular links between cell cycle control and the regulation of programmed cell death are largely unknown in plants. Here we studied the relationship between the cell cycle and elicitor-induced cell death using synchronized tobacco BY-2 cells. Flow cytometry and fluorescence microscopy of nuclear DNA, and RNA gel-blot analyses of cell cycle-related genes revealed that the proteinaceous elicitor cryptogein induced cell cycle arrest at the G1 or G2 phase before the induction of cell death. Furthermore, the patterns of cell death induction and defence-related genes were different in different phases of the cell cycle. Constitutive treatment with cryptogein induced cell cycle arrest and cell death at the G1 or G2 phase. With transient treatment for 2 h, cell cycle arrest and cell death were only induced by treatment with the elicitor during the S or G1 phase. By contrast, the elicitor-induced production of reactive oxygen species was observed during all phases of the cell cycle. These results indicate that although recognition of the elicitor signal is cell cycle-independent, the induction of cell cycle arrest and cell death depends on the phase of the cell cycle.     

Interrelation between salvage of purine nucleotides and protein synthesis in rat heart cells

The effect of transition from a respiring to a respiration-inhibited state on the rate of protein synthesis was investigated in glycolyzing, cultured rat heart cells. The rate was found to be significantly lower after blocking respiration, and it was further decreased by L-lactate. In contrast, pyruvate or phenazine methosulfate prevented the drop in the rate caused by lack of respiration. The changes in the respiratory state also affected the steady-state concentration of ATP, which varied in the same sense as the rate of protein synthesis. Pyruvate or phenazine methosulfate induced an increment in the concentration of ATP of respiration-inhibited cells. This increment could not be accounted for by more extensive phosphorylation of the available purine nucleotides, but required repletion of the pool by synthesis of purine nucleotides through the salvage pathway. Pyruvate and phenazine methosulfate were found to stimulate incorporation of labeled hypoxanthine into the purine nucleotide fraction in general, and into the nucleotide triphosphates in particular. Under similar incubation conditions an increase in the ATP/ADP ratio was also noted. The stimulatory effect of pyruvate on protein synthesis and on the cellular level of ATP was also observed in respiration-inhibited 3T6 cells and in human fibroblasts, but not in human fibroblasts deficient in the salvage enzyme, hypoxanthine-guanine-phosphoribosyltransferase. Based on the demonstrated influence of L-lactate, pyruvate, and phenazine methosulfate on the salvage synthesis of purine nucleotides [K. Ravid, P. Diamant, and Y. Avi-Dor, (1984) Arch. Biochem. Biophys. 229, 632-639] and on the present findings, the connection between protein synthesis and the salvage activity is discussed.     

Alterations in pyrimidine nucleotide metabolism as an early signal during the execution of programmed cell death in tobacco BY-2 cells

Changes in pyrimidine metabolism were investigated during programmed cell death (PCD) of tobacco BY-2 cells, induced by a simultaneous increase in the endogenous levels of nitric oxide (NO) and hydrogen peroxide. The de novo synthesis of pyrimidine nucleotides was estimated by following the metabolic fate of the (14)C-labelled orotic acid, whereas the rates of salvage and degradation pathways were studied by measuring the respective incorporation of (14)C-labelled uridine and uracil under different treatments. Nucleic acid metabolism was also examined using labelled thymidine as a marker. The results show that specific alterations in the balance of pyrimidine nucleotide synthesis, which include a decreased rate of salvage activity of uracil and uridine and increased salvage activity of thymidine, represent a metabolic switch that establishes proper cellular conditions for the induction of PCD. In particular, a reduction in the utilization of uracil for salvage products occurs very early during PCD, before the appearance of typical cytological features of the death programme, thus representing an early metabolic marker for PCD. These changes are strictly associated with PCD, since they do not occur if NO or hydrogen peroxide are increased individually, or if actinomycin, which inhibits the death programme, is added into the medium in the presence of NO and hydrogen peroxide. The possible roles of these fluctuations in pyrimidine metabolism on the cellular nucleotide pool are discussed in relation to the induction of cell death.     

Oligochitosan induces programmed cell death in tobacco suspension cells

Oligochitosan has been proved to trigger plant cell death. To gain some insights into the mechanisms of oligochitosan-induced cell death, the nature of oligochitosan-induced cell death and the role of calcium (Ca²⁺), nitric oxide (NO) and hydrogen peroxide (H₂O₂) were studied in tobacco suspension cells. Oligochitosan-induced cell death occurred in cytoplasmic shrinkage, phosphatidylserine externalization, chromatin condensation, TUNEL-positive nuclei, cytochrome c release and induction of programmed cell death (PCD)-related gene hsr203J, suggesting the activation of PCD pathway. Pretreatment cells with cyclosporin A, resulted in reducing oligochitosan-induced cytochrome c release and cell death, indicating oligochitosan-induced PCD was mediated by cytochrome c. In the early stage, cells undergoing PCD showed an immediate burst in free cytosolic Ca²⁺ ([Ca²⁺]_cyt) elevation, NO and H₂O₂ production. Further study showed that these three signals were involved in oligochitosan-induced PCD, while Ca²⁺ and NO played a negative role in this process by modulating cytochrome c release.     

Proteasome function is required for activation of programmed cell death in heat shocked tobacco Bright-Yellow 2 cells

To find out whether and how proteasome is involved in plant programmed cell death (PCD) we measured proteasome function in tobacco cells undergoing PCD as a result of heat shock (HS-PCD). Reactive oxygen species (ROS) production, cytochrome c levels and caspase-3-like protease activation were also measured in the absence or presence of MG132, a proteasome inhibitor. We show that proteasome activation occurs in early phase of HS-PCD upstream of the caspase-like proteases activation; moreover inhibition of proteasome function by MG132 results in prevention of PCD perhaps due to the prevention of ROS production, cytochrome c release and caspase-3-like protease activation.     

Cell death induced by sodium nitroprusside and hydrogen peroxide in tobacco BY-2 cell suspension

The interplay between nitric oxide (NO) and reactive oxygen species can lead to an induction of cell death in plants. The aim of our work was to find out if cyanide released from sodium nitroprusside (SNP; a donor of NO) could be involved in the cell death induction, which is triggered by SNP and H₂O₂. Cell suspension of Nicotiana tabacum L. (line BY-2) was treated with 0.5 mM SNP, 0.5 mM potassium ferricyanide (PFC; analogue of sodium nitroprusside which can not release NO) and/or by 0.5 mM glucose with 0.5 U cm⁻³ glucose oxidase (GGO; a donor system of H₂O₂). The cell death was induced only by combination of SNP and GGO. Thus cyanide released was not involved in the induction of cell death. However, SNP showed toxic effect because of decrease in activities of intracellular oxidoreductases and esterases. The cell death caused by SNP and GGO occurred within 12 h. During cell death either length or width of the cell increased. Central vacuole was formed in 20 to 40 % of cells. Most of the dead cells showed a condensed cytoplasm. Two hallmarks of programmed cell death (PCD), chromatin condensation and blebbing of nuclear periphery, were observed. However, oligonucleosomal fragmentation of DNA, another hallmark of PCD, was not detected.     

Balancing de novo synthesis and salvage of lipids by Leishmania amastigotes

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Changes in the de novo, salvage, and degradation pathways of pyrimidine nucleotides during tobacco shoot organogenesis.

Pyrimidine nucleotide metabolism was studied in tobacco callus cultured for 21days under shoot-forming (SF) and non-shoot-forming (NSF) conditions by following the metabolic fate of orotic acid, a precursor of the de novo pathway, and uridine and uracil, intermediates of the salvage and degradation pathways respectively. Nucleic acid synthesis was also investigated by measuring the incorporation of labeled thymidine into different cellular components. Our results indicate that with respect to nucleotide metabolism, the organogenic process in tobacco can be divided in two "metabolic phases": a de novo phase followed by a salvage phase. The initial stages of meristemoid formation during tobacco organogenesis (up to day 8) are characterized by a heavy utilization of orotic acid into nucleotides and nucleic acids. Utilization of this intermediate for the de novo synthesis of nucleotides, which is limited in NSF tissue, is mainly due to the activity of orotate phosphoribosyltransferase (OPRT), which increases in tissue cultured under SF conditions. After day 8, nucleotide synthesis during shoot growth seems to be mainly due to the salvage activity of both uridine and uracil. Both intermediates are preferentially utilized in SF tissue for the formation of nucleotides and nucleic acids through the activities of their respective salvage enzymes: uridine kinase (URK), and uracil phosphoribosyltransferase (UPRT). Metabolic studies on thymidine indicate that in SF tissue maximal nucleic acid synthesis occurs at day 4, in support of the initiation of meristemoid formation. Overall these results suggest that the organogenic process in tobacco is underlined by precise fluctuations in pyrimidine metabolism which delineate structural events culminating in shoot formation.     

Abnormal regulation of de novo purine synthesis and purine salvage in a cultured mouse T-cell lymphoma mutant partially deficient in adenylosuccinate synthetase.

The isolation and characterization of a mutant murine T-cell lymphoma (S49) with altered purine metabolism is described. This mutant, AU-100, was isolated from a mutagenized population of S49 cells by virtue of its resistance to 0.1 mM 6-azauridine in semisolid agarose. The AU-100 cells are resistant to adenosine mediated cytotoxicity but are extraordinarily sensitive to killing by guanosine. High performance liquid chromatography of AU-100 cell extracts has demonstrated that intracellular levels of GTP, IMP, and GMP are all elevated about 3-fold over those levels found in wild type cells. The AU-100 cells also contain an elevated intracellular level of pyrophosphoribosylphosphate (PPriboseP), which as in wild type cells is diminished by incubation of AU-100 cells with adenosine. However AU-100 cells synthesize purines de novo at a rate less than 35% of that found in wild type cells. In other growth rate experiments, the AU-100 cell line was shown to be resistant to 6-thioguanine and 6-mercaptopurine. Levels of hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) measured in AU-100 cell extracts, however, are 50-66% greater than those levels of HGPRTase found in wild type cell extracts. Nevertheless this mutant S49 cell line cannot efficiently incorporate labeled hypoxanthine into nucleotides since the salvage enzyme HGPRTase is inhibited in vivo. The AU-100 cell line was found to be 80% deficient in adenylosuccinate synthetase, but these cells are not auxotrophic for adenosine or other purines. The significant alterations in the control of purine de novo and salvage metabolism caused by the defect in adenylosuccinate synthetase are mediated by the resulting increased levels of guanosine nucleotides.     

Consequences of the salvage of purine compounds on the proliferation of rat T-lymphocytes with normal or inhibited purine de novo synthesis

We studied the ability of purine compounds to restore the proliferation of concanavalin-A-stimulated rat T-lymphocytes under conditions of purine de novo synthesis inhibition and, on the other hand, the inhibition by purine nucleosides of the response of these cells to a mitogenic stimulation under conditions of normal purine de novo synthesis. The use of 50 μM azaserine, a potent inhibitor of purine de novo synthesis, allowed us to define the physiologically active salvage pathways of purine bases, ribo- and deoxyribonucleosides in concanavalin-A-stimulated rat T-lymphocytes. Except for guanylic compounds, all purines completely restored cell proliferation at a concentration of 50 μM. Guanine, guanosine and 2′-deoxyguanosine at concentrations up to 500 μM did not allow us to restore more than 50% of the cell proliferation. In conditions of normal purine de novo synthesis, the addition of 1000 μM adenine, adenosine, 2′-deoxyadenosine or 100 μM 2′-deoxyguanosine inhibited rat T-lymphocyte proliferation. The differences between the degree of inhibition of cell proliferation could be explained only in part by the differences between the capacities of salvage of these compounds. Furthermore, the fact that 2′-deoxyguanosine toxicity was dependent and 2′-deoxyadenosine toxicity independent on the activation state of the cells provided more evidence that the biochemical mechanisms of inhibition of cell proliferation should be different for these two nucleosides.