GTP Depletion Research Articles

This Month in Gastroenterology

This Month in Gastroenterology

Induction of apoptosis by novel inosine monophosphate dehydrogenase, 3-hydrogenkwadaphnin

Today’s medical word is highly dependent on severel natural products (e.g. taxol) in fighting different kinds of cancer and in constantly working to found new compounds. In this respect, 3-hydrogenkwadaphnin is a new diterpene ester isolated from Dendrostellera lessertii (Thymelaceae). It has been previously shown that this new compound has high anti-tumor activity and the capability of arresting the growing leukemic cells at G1/S phase of their progression cycle, which finally leads to cell death. Further studies revealed 3-hydrogenkwadaphnin decreased both DNA synthesis and inosine monophosphate dehydrogenase (IMPDH) activity in K562 and HL-60 cells. Here we decided to interrogate the mechanism (s) and mode of cytotoxic effects of this compound. Inosine monophosphate dehydrogenase is the key regulatory and the rate-limiting enzyme of the de novo biosynthetic pathway of purines. Apparently, the activity of this enzyme significantly increases in tumor cells in proportion to the proliferation. We hypothesized that inhibition of IMPDH and consequently depletion of guanine nucleotide pool is responsible of cytotoxic effect with characteristic of apoptosis. HL-60 and K562 leukemia cells were treated with increasing doses of 3-hydrogenkwadaphnin. Thereafter, the mode of its cytotoxic action was determined by Acridine orange/Ethedium bromide double staining, DNA fragmentation, sub-G1 flow cytometry and electron microscopic. Involvement of guanine nucleotide pool was tested by co-provision of guanosine and deoxyguanosine that replead GTP and dGTP pool size, respectively. 3-hydrogenkwadaphnin at low doses (10 nM) enhanced fragment of the apoptotic HL-60 cells within 72 hours of treatment. Higher doses (25 nM) led to rapid induction of apoptosis, which could be detected as early as 24 h. The majority (85%) of the treated HL-60 cells died by apoptosis, 72 hours after exposure to 3-hydrogenkwadaphnin. Althought K562 cells are generally more resistant to a variety of apoptotic stimuli, to our surprising 3-hydrogenkwadaphnin was equally effective in this cell line as compared to HL-60. 3-hydrogenkwadaphnin-induced apoptosis was almost totally prevented by supplementation with guanosine, but not with adenosine or deoxyguanosine, indicating the specific effect of 3-hydrogenkwadaphnin in reducing the pool size of GTP. The results indicate that 3-hydrogenkwadaphnin decreases GTP pool size and depletion of this pool, and not dGTP, is probably the cause of cell death through apoptosis. Induction of apoptosis is a major goal of chemotherapy. GTP depletion induced apoptosis is selective for tumor cells; thereby 3-hydrogenkwadaphnin could be presented as a powerful drug for treatment of cancer, especially leukemia.

Cellular IMPDH enzyme activity is a potential target for the inhibition of Chikungunya virus replication and virus induced apoptosis in cultured mammalian cells

Inosine monophosphate dehydrogenase (IMPDH) catalyzes an essential step in the de novo biosynthesis of guanine nucleotide, namely, the conversion of IMP to XMP. The depletion of the intracellular GTP and dGTP pools is the major event occurring in the cells exposed to the inhibitors such as mycophenolic acid. The present study was undertaken with an objective to assess the antiviral potential of mycophenolic acid (MPA) against Chikungunya virus via inhibition of IMPDH enzyme in Vero cells. The inhibitory potential of MPA on CHIKV replication was assessed by virus inhibition assay (cytopathic effect, immunofluorescence), virus yield reduction assay and cell viability assay. Inhibition of virus induced apoptosis was analyzed by Hoechst staining, DNA fragmentation, immunoblotting of Caspase-3, PARP and Bcl-2. Percentage apoptotic cell population was determined by flow cytometry. Total genome infectivity was determined by analyzing the ratio of total infectious viral particles to the genome copy number. Non-toxic concentration of MPA (10 μM) reduced ≥99.9% CHIKV titre in Vero cells. MPA via depletion of substrate for polymerase (GTP), inhibited CHIKV induced apoptosis. By limiting the rate of de novo synthesis of guanosine nucleotide, MPA could apparently block the formation of the CHIKV progeny. The antiviral activity of MPA against Chikungunya virus is mediated through depletion of GTP pool via inhibition of IMPDH as demonstrated by Immunoblotting and different microscopic analysis.

A novel nucleoside analog, 1-β- d-ribofuranosyl-3-ethynyl-[1,2,4]triazole (ETAR), exhibits efficacy against a broad range of flaviviruses in vitro

Antiviral therapies are urgently needed to control emerging flaviviruses such as dengue, West Nile, and yellow fever. Ribavirin (RBV) has shown activity against flaviviruses in cultured cells, but efficacy in animal models has generally been poor. In a preliminary screen of novel, synthetic 1-β- d-ribofuranosyl-azole analogs, two compounds, 1-β- d-ribofuranosyl-3-ethynyl-[1,2,4]triazole (ETAR) and 1-β- d-ribofuranosyl-4-ethynyl-[1,3]imidazole (IM18), significantly reduced the replication of dengue virus serotype 2 (DENV-2) in cultured Vero cells. In the current study we demonstrated that the effective concentration 50 (EC 50) of ETAR for DENV-2 is substantially lower than both IM18 and RBV. Moreover, ETAR reduced the replication of five additional flaviviruses, including DENV serotypes 1, 3 and 4, Langat virus and Modoc virus, ≥1000-fold relative to untreated controls. Addition of exogenous guanosine to DENV-2 infected cells negated the antiviral effects of both RBV and ETAR, indicating that GTP depletion is a major mechanism of action for both drugs. ETAR represents a promising drug candidate for the treatment of flavivirus infections.

641 DEVELOPMENT OF IN VITRO AND CELL-BASED ASSAYS FOR ASSESSING NUCLEOTIDE BIOSYNTHESIS INHIBITION

[1] D.G. Streeter, J. T. Witkowski, G.P. Khare, R.W. Sidwell,r;j. Bauer, R.K. Robins, L. N. Simon (1973) Mechanism of Action of 1-i3-DRibofuranosyl-1,2,4-Triazole-3-Carboxamide (Virazole), A New Broad-Spectrum Antiviral Agent Proc. Nat. Acad. Sci. USA Vol. 70, No. 4, pp. 1174-1178 [2] R. C. Willis, D. A. Carson, and J. E. Seegmiller (1973) Adenosine Kinase Initiates the Major Route of Ribavirin Activation in a Cultured Human Cell Line. PNAS USA, 75: 3042-3044. [3] Wu JZ, Larson G, Walker H, Shim JH, Hong Z. Phosphorylation of ribavirin and viramidine by adenosine kinase and cytosolic 5'nucleotidase II: Implications for ribavirin metabolism in erythrocytes. (2005) Antimicrob Agents Chemother. 49(6):2164-71 References Background Numerous nucleoside analogues (NAs) are currently used to treat viral hepatitis. They are usually designed to inhibit one viral target. This remains in contrast with the observation that ribavirin (1-β-D-ribofuranosyl1,2,4-triazole-3-carboxamide), a purine nucleoside analogue currently used as a part of bi-therapy of hepatitis C infection, has multiple modes of action: (i) depletion of intracellular GTP pools by inhibition of the cellular IMPDH, (ii) inhibition of viral polymerase activity by the 5’-triphosphate metabolite of ribavirin, (iii) induction of error catastrophe as a result of accumulation of mutations in the viral genome. Even if no direct relationship between ribavirin antiviral action and IMPDH inhibition has been demonstrated, the depletion of cellular GTP should result in an increased frequency of ribavirin triphosphate incorporation by viral polymerase due to lower intracellular concentration of its natural competitor.

642 CHARACTERISATION OF HEPATITIS C VIRUS ENTRY AND RNA REPLICATION IN CELLS OF CNS ORIGIN

(ii) inhibition of viral polymerase activity by the 5’-triphosphate metabolite of ribavirin, (iii) induction of error catastrophe as a result of accumulation of mutations in the viral genome. Even if no direct relationship between ribavirin antiviral action and IMPDH inhibition has been demonstrated, the depletion of cellular GTP should result in an increased frequency of ribavirin triphosphate incorporation by viral polymerase due to lower intracellular concentration of its natural competitor. Aims: Relation between therapeutic potential of a nucleoside analogue and its anti-metabolite action remains difficult to demonstrate mainly because of the lack of investigation tools. The purpose of this study was to develop a range of assays to reveal nucleotide biosynthesis inhibition. Results: For a rapid evaluation of new nucleoside analogues as IMPDH inhibitors, we have developed an in vitro enzymatic assay where the synthesis of monophosphorylated form of nucleoside analogue (NA-MP) is provided by cloned human nucleoside kinases, and NA-MP is immediately tested for inhibition of human recombinant IMPDH. This assay has been validated with nucleoside analogues ribavirin and mizoribine. We have also developed original cell-based analytical approach in which 27 cellular riboand deoxyribonucleotides are extracted from cultured cells, separated by ion-pairing chromatography and quantified. This cellular assay, validated with several NA (ribavirin, aracytidine, gemcitabine) and known anti-metabolites (mycophenolic acid, leflunomide, hydroxyurea), provides a powerful tool for studying the effect of new nucleoside analogues on whole spectra of cellular purine and pyrimidine deoxyand ribonucleotides. In addition, in regards with new antiviral molecules identified in HCV cell culture systems, our cellular assay allows to distinguish the molecules that directly acts on the viral proteins from others that inhibits the cell nucleotide biosynthesis.

Mycophenolic Acid Activation of p53 Requires Ribosomal Proteins L5 and L11

Mycophenolate mofetil (MMF), a prodrug of mycophenolic acid (MPA), is widely used as an immunosuppressive agent. MPA selectively inhibits inosine monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme for the de novo synthesis of guanine nucleotides, leading to depletion of the guanine nucleotide pool. Its chemotherapeutic effects have been attributed to its ability to induce cell cycle arrest and apoptosis. MPA treatment has also been shown to induce and activate p53. However, the mechanism underlying the p53 activation pathway is still unclear. Here, we show that MPA treatment results in inhibition of pre-rRNA synthesis and disruption of the nucleolus. This treatment enhances the interaction of MDM2 with L5 and L11. Interestingly, knockdown of endogenous L5 or L11 markedly impairs the induction of p53 and G(1) cell cycle arrest induced by MPA. These results suggest that MPA may trigger a nucleolar stress that induces p53 activation via inhibition of MDM2 by ribosomal proteins L5 and L11.

Endoplasmic reticulum-associated degradation of mutant CFTR requires a guanine nucleotide-sensitive step

Proteasome degradation of endoplasmic reticulum (ER)-misfolded proteins requires retrograde transport from ER to the cytosol. To date, it is not clear whether this event constitutes the exclusive ER degradation process for non-native membrane proteins. Here we describe the role of GTP in the degradation of ΔF508-CFTR and the α subunit of the T-cell receptor (TCRα), representative misfolded ER membrane proteins. Selective intracellular GTP depletion extended the ΔF508-CFTR half-life sixfold, whereas ATP depletion accelerated its turnover and inhibited only 80% of the proteasome activity that was not affected by GTP depletion. AlF4−, a well-known inhibitor of heterotrimeric G proteins, but not of AlF3, delayed the mutant CFTR turnover in vivo, in semi-intact cells and in ER-enriched microsomes, without affecting ER to Golgi cargo transport. ΔF508-CFTR degradation was also inhibited by alkaline stripping of ER-associated membrane proteins. We propose that at the ER, GTP may participate in the disposal of misfolded membrane proteins through activation of heterotrimeric G proteins.

Interferons and Ribavirin Effectively Inhibit Norwalk Virus Replication in Replicon-Bearing Cells

The development of effective therapies for noroviral gastroenteritis has been hampered by the absence of a cell culture system. Recently, we reported the generation of Norwalk virus (NV) replicon-bearing cells in BHK21 and Huh-7 cells and demonstrated that alpha interferon (IFN-alpha) effectively inhibited the replication of NV in these cells. In continuing studies for screening potential antinoroviral agents, we tested IFN-gamma and ribavirin for their effects on NV replication in the cells. Like IFN-alpha, IFN-gamma inhibited the replication of NV in the replicon-bearing cells, showing the reduction of the NV genome and proteins in a dose-dependent manner. The effective dose for reducing 50% (ED(50)) of the NV genome and protein was calculated to be approximately 40 units/ml. When ribavirin was applied to the cells, it effectively reduced the NV genome and protein with the ED(50) calculated as approximately 40 microM. The combination of IFN-alpha and ribavirin showed additive effects on the inhibition of NV replication. With the addition of guanosine to the ribavirin treatment, moderately reversed antiviral effects were observed, suggesting that the ribavirin effect may be associated with the depletion of GTP in the cells. Sequencing analysis of the conserved polymerase regions of NV in the ribavirin-treated (100 microM) and nontreated groups showed that the mutation rates were similar and indicated that ribavirin did not induce catastrophic mutations. The NV replicon-bearing cells provide an excellent tool for screening potential antinoroviral agents, and our results indicated that IFNs and ribavirin may be good therapeutic options for noroviral gastroenteritis.

Ribavirin inhibits angiogenesis by tetrahydrobiopterin depletion

Ribavirin is a broad-spectrum antiviral drug that is used to treat hepatitis C virus (HCV)-infected patients. The virological response after ribavirin treatment appears to be insufficient to fully explain ribavirin-induced beneficial effects. Angiogenesis plays a pathogenic role in HCV-induced liver damage. Here, we investigated the influence of therapeutic ribavirin concentrations on angiogenesis. Ribavirin inhibited endothelial cell tube formation in vitro and vessel formation in the chick chorioallantoic membrane assay in vivo. Ribavirin inhibits inosine monophosphate dehydrogenase, which causes depletion of cellular GTP and in turn reduction of cellular tetrahydrobiopterin levels. The availability of tetrahydrobiopterin limits NO production by endothelial NO synthase. Ribavirin reduced levels of tetrahydrobiopterin (as revealed by HPLC), NO (as revealed by electron spin resonance spectroscopy), and cGMP (as revealed by RIA) in endothelial cells. Addition of tetrahydrobiopterin or NO prevented ribavirin-induced tube formation inhibition. In conclusion, angiogenesis inhibition by ribavirin has not been described before. This inhibition may contribute to ribavirin-induced pharmacological effects including adverse events.

A Novel Lysine-rich Domain and GTP Binding Motifs Regulate the Nucleolar Retention of Human Guanine Nucleotide Binding Protein, GNL3L

A variety of G-proteins and GTPases are known to be involved in nucleolar function. We describe here a new evolutionarily conserved putative human GTPase, guanine nucleotide binding protein-like 3-like (GNL3L). Genes encoding proteins related to GNL3L are present in bacteria and yeast to metazoa and suggests its critical role in development. Conserved domain search analysis revealed that the GNL3L contains a circularly permuted G-motif described by a G5-G4-G1-G2-G3 pattern similar to the HSR1/MMR1 GTP-binding protein subfamily. Highly conserved and critical residues were identified from a three-dimensional structural model obtained for GNL3L using the crystal structure of an Ylqf GTPase from Bacillus subtilis. We demonstrate here that GNL3L is transported into the nucleolus by a novel lysine-rich nucleolar localization signal (NoLS) residing within 1–50 amino acid residues. NoLS identified here is necessary and sufficient to target the heterologous proteins to the nucleolus. We show for the first time that the lysine-rich targeting signal interacts with the nuclear transport receptor, importin-beta and transports GNL3L into the nucleolus. Interestingly, depletion of intracellular GTP blocks GNL3L accumulation into the nucleolar compartment. Furthermore, mutations within the G-domains alter the GTP binding ability of GNL3L and abrogate wild-type nucleolar retention even in the presence of functional NoLS, suggesting that the efficient nucleolar retention of GNL3L involves activities of both basic NoLS and GTP-binding domains. Collectively, these data suggest that GNL3L is composed of distinct modules, each of which plays a specific role in molecular interactions for its nucleolar retention and subsequent function(s) within the nucleolus.

Effects of a natural extract from Mangifera indica L, and its active compound, mangiferin, on energy state and lipid peroxidation of red blood cells

Following oxidative stress, modifications of several biologically important macromolecules have been demonstrated. In this study we investigated the effect of a natural extract from Mangifera indica L (Vimang), its main ingredient mangiferin and epigallocatechin gallate (EGCG) on energy metabolism, energy state and malondialdehyde (MDA) production in a red blood cell system. Analysis of MDA, high energy phosphates and ascorbate was carried out by high performance liquid chromatography (HPLC). Under the experimental conditions, concentrations of MDA and ATP catabolites were affected in a dose-dependent way by H 2O 2. Incubation with Vimang (0.1, 1, 10, 50 and 100 μg/mL), mangiferin (1, 10, 100 μg/mL) and EGCG (0.01, 0.1, 1, 10 μM) significantly enhances erythrocyte resistance to H 2O 2-induced reactive oxygen species production. In particular, we demonstrate the protective activity of these compounds on ATP, GTP and total nucleotides (NT) depletion after H 2O 2-induced damage and a reduction of NAD and ADP, which both increase because of the energy consumption following H 2O 2 addition. Energy charge potential, decreased in H 2O 2-treated erythrocytes, was also restored in a dose-dependent way by these substances. Their protective effects might be related to the strong free radical scavenging ability described for polyphenols.

3-Hydrogenkwadaphnin fromDendrostellera lessertiiInduces Differentiation and Apoptosis in HL-60 Cells

3-Hydrogenkwadaphnin (3-HK) is a new diterpene ester, recently isolated from the leaves of Dendrostellera lessertii with potent anti-tumoral and anti-metastatic activities. Herein, we report that 3-HK induces differentiation and apoptosis in HL-60 cells. The drug at 2.5 - 40 nM inhibited proliferation of HL-60 cells after 24 - 96 h of treatment. Cell viability was also decreased by 57 % after 96 h treatment with the drug. NBT reducing assay and phagocytic activity revealed that the inhibition of proliferation is associated with differentiation especially toward macrophages-like morphology. Indeed, the drug at 2.5 - 10 nM induced differentiation by 5 - 49 % in HL-60 cells. Acridine orange/ethidium bromide (AO/EtBr) double staining and DNA fragmentation assays revealed that apoptosis occurred after differentiation of HL-60 cells. Guanosine at 50 microM decreased the apoptotic cell death and the differentiation caused by the drug. Therefore, GTP depletion, as a result of inhibition of inosine monophosphate dehydrogenase (IMPDH), is considered as one of the main reasons for differentiation and apoptotic cell death by this new drug.

Light-Dependent Redistribution of Arrestin in Vertebrate Rods Is an Energy-Independent Process Governed by Protein-Protein Interactions

In rod photoreceptors, arrestin localizes to the outer segment (OS) in the light and to the inner segment (IS) in the dark. Here, we demonstrate that redistribution of arrestin between these compartments can proceed in ATP-depleted photoreceptors. Translocation of transducin from the IS to the OS also does not require energy, but depletion of ATP or GTP inhibits its reverse movement. A sustained presence of activated rhodopsin is required for sequestering arrestin in the OS, and the rate of arrestin relocalization to the OS is determined by the amount and the phosphorylation status of photolyzed rhodopsin. Interaction of arrestin with microtubules is increased in the dark. Mutations that enhance arrestin-microtubule binding attenuate arrestin translocation to the OS. These results indicate that the distribution of arrestin in rods is controlled by its dynamic interactions with rhodopsin in the OS and microtubules in the IS and that its movement occurs by simple diffusion.

Role of human nucleoside transporters in the cellular uptake of two inhibitors of IMP dehydrogenase, tiazofurin and benzamide riboside.

Benzamide riboside (BR) and tiazofurin (TR) are converted to analogs of NAD that inhibit IMP dehydrogenase (IMPDH), resulting in cellular depletion of GTP and dGTP and inhibition of proliferation. The current work was undertaken to identify the human nucleoside transporters involved in cellular uptake of BR and TR and to evaluate their role in cytotoxicity. Transportability was examined in Xenopus laevis oocytes and Saccharomyces cerevisiae that produced individual recombinant human concentrative nucleoside transporter (CNT) and equilibrative nucleoside transporter (ENT) types (hENT1, hENT2, hCNT1, hCNT2, or hCNT3). TR was a better permeant than BR with a rank order of transportability in oocytes of hCNT3 >> hENT1 > hENT2 > hCNT2 >> hCNT1. The concentration dependence of inhibition of [(3)H]uridine transport in S. cerevisiae by TR exhibited lower K(i) values than BR: hCNT3 (5.4 versus 226 microM), hENT2 (16 versus 271 microM), hENT1 (57 versus 168 microM), and hCNT1 (221 versus 220 microM). In cytotoxicity experiments, BR was more cytotoxic than TR to cells that were either nucleoside transport-defective or -competent, and transport-competent cells were more sensitive to both drugs. Exposure to nitrobenzylmercaptopurine ribonucleoside conferred resistance to BR and TR cytotoxicity to hENT1-containing CEM cells, thereby demonstrating the importance of transport capacity for manifestation of cytoxicity. A breast cancer cell line with mutant p53 exhibited 9-fold higher sensitivity to BR than the otherwise similar cell line with wild-type p53, suggesting that cells with mutant p53 may be potential targets for IMPDH inhibitors. Further studies are warranted to determine whether this finding can be generalized to other cell types.

P53-independent induction of p21(waf1/cip1) contributes to the activation of caspases in GTP-depletion-induced apoptosis of insulin-secreting cells.

We investigated the role of some key regulators of cell cycle in the activation of caspases during apoptosis of insulin-secreting cells after sustained depletion of GTP by a specific inosine 5'-monophosphate dehydrogenase inhibitor, mycophenolic acid (MPA). p21(Waf1/Cip1) was significantly increased following MPA treatment, an event closely correlated with the time course of caspase activation under the same conditions. MPA-induced p21(Waf1/Cip1) was not mediated by p53, since p53 mass was gradually reduced over time of MPA treatment. The increment of p21(Waf1/Cip1) by MPA was further enhanced in the presence of a pan-caspase inhibitor, indicating that the increased p21(Waf1/Cip1) may occur prior to caspase activation. This notion of association of p21(Waf1/Cip1) accumulation with caspase activation and apoptosis was substantiated by using mimosine, a selective p21(Waf1/Cip1) inducer independent of p53. Mimosine, like MPA, also increased p21(Waf1/Cip1), promoted apoptosis and simultaneously increased the activity of caspases. Furthermore, knocking down of p21(Waf1/Cip1) transfection of siRNA duplex inhibited caspase activation and apoptosis due to GTP depletion. In contrast to p21(Waf1/Cip1), a reduction in p27(Kip1) occurred in MPA-treated cells. These results indicate that p21(Waf1/Cip1) may act as an upstream signal to block mitogenesis and activate caspases which in turn contribute to induction of apoptosis.

Essential Cell Division Protein FtsZ Assembles into One Monomer-thick Ribbons under Conditions Resembling the Crowded Intracellular Environment

Experimental conditions that simulate the crowded bacterial cytoplasmic environment have been used to study the assembly of the essential cell division protein FtsZ from Escherichia coli. In solutions containing a suitable concentration of physiological osmolytes, macromolecular crowding promotes the GTP-dependent assembly of FtsZ into dynamic two-dimensional polymers that disassemble upon GTP depletion. Atomic force microscopy reveals that these FtsZ polymers adopt the shape of ribbons that are one subunit thick. When compared with the FtsZ filaments observed in vitro in the absence of crowding, the ribbons show a lag in the GTPase activity and a decrease in the GTPase rate and in the rate of GTP exchange within the polymer. We propose that, in the crowded bacterial cytoplasm under assembly-promoting conditions, the FtsZ filaments tend to align forming dynamic ribbon polymers. In vivo these ribbons would fit into the Z-ring even in the absence of other interactions. Therefore, the presence of mechanisms to prevent the spontaneous assembly of the Z-ring in non-dividing cells must be invoked.

Role of tissue transglutaminase in GTP depletion-induced apoptosis of insulin-secreting (HIT-T15) cells

The role of tissue transglutaminase (tTG), a calcium-dependent and GTP-modulated enzyme, in apoptotic death induced by GTP depletion in islet β-cells was investigated. GTP depletion and apoptosis were induced by mycophenolic acid (MPA) in insulin-secreting HIT-T15 cells. MPA treatment increased in situ tTG activity (but not protein levels) in a dose- and time-dependent manner in parallel with the induction of apoptosis. MPA-induced increases of both tTG activity and apoptosis were entirely blocked by co-provision of guanosine but not adenosine. MPA-enhanced tTG activity could be substantially reduced by co-exposure to monodansylcadaverine or putrescine (tTG inhibitors), and largely blocked by lowering free Ca 2+ concentrations in the culture medium. However, MPA-induced cell death was either not changed or was only slightly reduced under these conditions. By contrast, a pan-caspase inhibitor (Z-VAD-FMK) entirely prevented apoptosis induced by MPA, but did not block the enhanced tTG activity, indicating that GTP depletion can induce apoptosis and activate tTG either independently or as part of a cascade of events involving caspases. Importantly, the morphological changes accompanying apoptosis could be markedly prevented by tTG inhibitors. These findings suggest that the effect of the marked increase in tTG activity in GTP depletion-induced apoptosis of insulin-secreting cells may be restricted to some terminal morphological changes.

Rho GTPases show differential sensitivity to nucleotide triphosphate depletion in a model of ischemic cell injury.

Rho GTPases are critical for actin cytoskeletal regulation, and alterations in their activity may contribute to altered cytoskeletal organization that characterizes many pathological conditions, including ischemia. G protein activity is a function of the ratio of GTP-bound (active) to GDP-bound (inactive) protein, but the effect of altered energy metabolism on Rho protein activity has not been determined. We used antimycin A and substrate depletion to induce depletion of intracellular ATP and GTP in the kidney proximal tubule cell line LLC-PK10 and measured the activity of RhoA, Rac1, and Cdc42 with GTPase effector binding domains fused to glutathione S-transferase. RhoA activity decreased in parallel with the concentration of ATP and GTP during depletion, so that by 60 min there was no detectable RhoA-GTP, and recovered rapidly when cells were returned to normal culture conditions. Dissociation of the membrane-actin linker ezrin, a target of RhoA signaling, from the cytoskeletal fraction paralleled the decrease in RhoA activity and was augmented by treatment with the Rho kinase inhibitor Y27632. The activity of Cdc42 did not decrease significantly during depletion or recovery. Rac1 activity decreased moderately to a minimum at 30 min of depletion but then increased from 30 to 90 min of depletion, even as ATP and GTP levels continued to fall. Our data are consistent with a principal role for RhoA in cytoskeletal reorganization during ischemia and demonstrate that the activity of Rho GTPases can be maintained even at low GTP concentrations.

P53 mediates the apoptotic response to GTP depletion after renal ischemia-reperfusion: protective role of a p53 inhibitor.

Ischemic injury to the kidney is characterized in part by nucleotide depletion and tubular cell death in the form of necrosis or apoptosis. GTP depletion was recently identified as an important inducer of apoptosis during chemical anoxia in vitro and ischemic injury in vivo. It has also been shown that GTP salvage with guanosine prevented apoptosis and protected function. This study investigates the role of p53 in mediating the apoptotic response to GTP depletion. Male Sprague-Dawley rats underwent bilateral renal artery clamp for 30 min followed by reperfusion. p53 protein levels increased significantly in the medulla over 24 h post-ischemia. The provision of guanosine inhibited the increase in p53. Pifithrin-alpha, a specific inhibitor of p53, mimicked the effects of guanosine. It had no effect on necrosis, yet it prevented apoptosis and protected renal function. Pifithrin-alpha was protective when given up to 14 h after the ischemic insult. The effects of pifithrin-alpha on p53 included inhibition of transcriptional activation of downstream p53 targets like p21 and Bax and inhibition of p53 translocation to the mitochondria. Similar results were obtained in cultured renal tubular cells. It is concluded that p53 is an important mediator of apoptosis during states of GTP depletion. Inhibitors of p53 should be considered in the treatment of ischemic renal injury.