Neuropathy-associated histidyl-tRNA synthetase variants attenuate protein synthesis in vitro and disrupt axon outgrowth in developing zebrafish.

Charcot-Marie-Tooth disease (CMT) encompasses a set of genetically and clinically heterogeneous neuropathies characterized by length-dependent dysfunction of the peripheral nervous system. Mutations in over 80 diverse genes are associated with CMT, and aminoacyl-tRNA synthetases (ARS) constitute a large gene family implicated in the disease. Despite considerable efforts to elucidate the mechanistic link between ARS mutations and the CMT phenotype, the molecular basis of the pathology is unknown. In this work, we investigated the impact of three CMT-associated substitutions (V155G, Y330C, and R137Q) in the cytoplasmic histidyl-tRNA synthetase (HARS1) on neurite outgrowth and peripheral nervous system development. The model systems for this work included a nerve growth factor-stimulated neurite outgrowth model in rat pheochromocytoma cells (PC12), and a zebrafish line with GFP/red fluorescent protein reporters of sensory and motor neuron development. The expression of CMT-HARS1 mutations led to attenuation of protein synthesis and increased phosphorylation of eIF2α in PC12 cells and was accompanied by impaired neurite and axon outgrowth in both models. Notably, these effects were phenocopied by histidinol, a HARS1 inhibitor, and cycloheximide, a protein synthesis inhibitor. The mutant proteins also formed heterodimers with wild-type HARS1, raising the possibility that CMT-HARS1 mutations cause disease through a dominant-negative mechanism. Overall, these findings support the hypothesis that CMT-HARS1 alleles exert their toxic effect in a neuronal context, and lead to dysregulated protein synthesis. These studies demonstrate the value of zebrafish as a model for studying mutant alleles associated with CMT, and for characterizing the processes that lead to peripheral nervous system dysfunction.

Activating transcription factor 4 mediates up-regulation of alanine aminotransferase 2 gene expression under metabolic stress.

Alanine aminotransferase (ALT) provides a molecular link between carbohydrate and amino acid metabolism. In humans, two ALT isoforms have been characterized: ALT1, cytosolic, and ALT2, mitochondrial. To gain insight into the transcriptional regulation of the ALT2 gene, we cloned and characterized the human ALT2 promoter. 5'-deletion analysis of ALT2 promoter in transiently transfected HepG2 cells and site-directed mutagenesis allowed us to identify ATF4 as a new factor involved in the transcriptional regulation of ALT2 expression. Quantitative RT-PCR assays showed that the metabolic stressors histidinol and tunicamycin increased ATF4 levels and up-regulated ALT2 in HepG2 and Huh7 cells. Consistently, knock-down of ATF4 decreased ALT2 mRNA levels in HepG2 and Huh-7 cells. Moreover, ATF4 silencing prevented the activating effect of histidinol and tunicamycin on ATF4 and ALT2 expression. Our findings point to ALT2 as an enzyme involved in the metabolic adaptation of the cell to stress.

Elevated cJUN expression and an ATF/CRE site within the ATF3 promoter contribute to activation of ATF3 transcription by the amino acid response.

Mammalian cells respond to amino acid deprivation through multiple signaling pathways referred to as the amino acid response (AAR). Transcription factors mediate the AAR after their activation by several mechanisms; examples include translational control (activating transcription factor 4, ATF4), phosphorylation (p-cJUN), and transcriptional control (ATF3). ATF4 induces ATF3 transcription through a promoter-localized C/EBP-ATF response element (CARE). The present report characterizes an ATF/CRE site upstream of the CARE that also contributes to AAR-induced ATF3 transcription. ATF4 binds to the ATF/CRE and CARE sequences and both are required for a maximal response to ATF4 induction. ATF3, which antagonizes ATF4 and represses its own gene, also exhibited binding activity to the ATF/CRE and CARE sequences. The AAR resulted in elevated total cJUN and p-cJUN protein levels and both forms exhibited binding activity to the ATF/CRE and CARE ATF3 sequences. Knockdown of AAR-enhanced cJUN expression blocked induction of the ATF3 gene and mutation of either the ATF/CRE or the CARE site prevented the cJUN-dependent increase in ATF3-driven luciferase activity. The results indicate that both increased cJUN and the cis-acting ATF/CRE sequence within the ATF3 promoter contribute to the transcriptional activation of the gene during the AAR.

Targeting of the Brucella suis virulence factor histidinol dehydrogenase by histidinol analogues results in inhibition of intramacrophagic multiplication of the pathogen.

Brucella suis histidinol dehydrogenase (HDH) can be efficiently targeted by substrate analogues. The growth of this pathogen in minimal medium was inhibited and the multiplication in human macrophages was totally abolished in the presence of the drugs. These effects have been shown to be correlated with the previously described inhibition of Brucella HDH activity.

A simple, informative, and quantitative flow cytometric method for assessing apoptosis in cultured cells.

Described herein is a method for assessing apoptosis in tissue culture cells that is facile, highly informative, readily quantifiable, and generally applicable. As in conventional DNA-based flow cytometric analysis, the approach utilizes fixed, propidium iodide-stained cells. However, this particular application employs correlated two-parameter analyses of log(10)DNA fluorescence signals versus log(10) side-scatter (SS) signals of cells undergoing apoptosis. The features and the advantages of this approach, which provides substantially more information than is otherwise available from conventional analysis, are demonstrated in experiments monitoring the effects of the antineoplastic agents cisplatinum (cisP) and camptothecin (CAM) on a variety of cultured cell types, including epithelial cells (SW480; human colon carcinoma), fibroblasts (rat2 and 3T3; normal fibroblast lines), and cells of myeloid origin (CCRF-CEM; human leukemia). The utility of the technique is demonstrated further in a series of experiments with the histidine analogue L-histidinol. These experiments reveal that L-histidinol is pro-apoptotic in CCRF-CEM cells, accentuates markedly the apoptotic response otherwise elicited by CAM in murine B16f10 melanoma cells and inhibits CAM-induced apoptosis in normal 3T3 fibroblasts.

Assessment of cell-signaling pathways in the regulation of mammalian target of rapamycin (mTOR) by amino acids in rat adipocytes.

Enhanced phosphorylation of the ribosomal protein s6 kinase, p70(s6k), and the translational repressor, 4E-BP1, are associated with either insulin-induced or amino acid-induced protein synthesis. Hyperphosphorylation of p70(s6k) and 4E-BP1 in response to insulin or amino acids is mediated through the mammalian target of rapamycin (mTOR). In several cell lines, mTOR or its downstream targets can be regulated by phosphatidylinositol (PI) 3-kinase; protein kinases A, B, and C; heterotrimeric G-proteins; a PD98059-sensitive kinase or calcium; as well as by amino acids. Regulation by amino acids appears to involve detection of levels of charged t-RNA or t-RNA synthetase activity and is sensitive to inhibition by amino acid alcohols. In the present article, however, we show that the rapamycin-sensitive regulation of 4E-BP1 and p70(s6k) in freshly isolated rat adipocytes is not inhibited by either L-leucinol or L-histidinol. This finding is in agreement with other recent studies from our laboratory suggesting that the mechanism by which amino acids regulate mTOR in freshly isolated adipocytes may be different than the mechanism found in a number of cell lines. Therefore we investigated the possible role of growth factor-regulated and G-protein-regulated signaling pathways in the rapamycin-sensitive, amino acid alcohol-insensitive actions of amino acids on 4E-BP1 phosphorylation. We found, in contrast to previously published results using 3T3-L1 adipocytes or other cell lines, that the increase in 4E-BP1 phosphorylation promoted by amino acids was insensitive to agents that regulate protein kinase A, mobilize calcium, or inhibit protein kinase C. Furthermore, amino acid-induced 4E-BP1 phosphorylation was not blocked by pertussis toxin nor was it mimicked by the G-protein agonists fluoroaluminate or MAS-7. However, amino acids failed to activate either PI 3-kinase, protein kinase B, or mitogen-activated protein kinase and failed to promote tyrosine phosphorylation of cellular proteins, similar to observations made using cell lines. In summary, amino acids appear to use an amino acid alcohol-insensitive mechanism to regulate mTOR in freshly isolated adipocytes. This mechanism is independent of cell-signaling pathways implicated in the regulation of mTOR or its downstream targets in other cells. Overall, our study emphasizes the need for caution when extending results obtained using established cell lines to the differentiated nondividing cells found in most tissues.

Histidinol phosphate phosphatase, catalyzing the penultimate step of the histidine biosynthesis pathway, is encoded by ytvP (hisJ) in Bacillus subtilis.

The deduced product of the Bacillus subtilis ytvP gene is similar to that of ORF13, a gene of unknown function in the Lactococcus lactis histidine biosynthesis operon. A B. subtilis ytvP mutant was auxotrophic for histidine. The only enzyme of the histidine biosynthesis pathway that remained uncharacterized in B. subtilis was histidinol phosphate phosphatase (HolPase), catalyzing the penultimate step of this pathway. HolPase activity could not be detected in crude extracts of the ytvP mutant, while purified glutathione S-transferase-YtvP fusion protein exhibited strong HolPase activity. These observations demonstrated that HolPase is encoded by ytvP in B. subtilis and led us to rename this gene hisJ. Together with the HolPase of Saccharomyces cerevisiae and the presumed HolPases of L. lactis and Schizosaccharomyces pombe, HisJ constitutes a family of related enzymes that are not homologous to the HolPases of Escherichia coli, Salmonella typhimurium, and Haemophilus influenzae.

Effects of L-histidinol on the antitumour activity and acute cardiotoxicity of doxorubicin in mice.

L-Histidinol (LHL), a structural analogue of the essential amino acid l-histidine, can improve the therapeutic index of antimetabolites and alkylating agents. The objective of the study was to determine whether LHL would modulate the antitumour activity and acute cardiotoxicity of the anthracycline antibiotic, doxorubicin (DOX). LHL (1.0 mM) potentiated the cytotoxicity of DOX (0.05-0.8 microg ml-1) in cultured Ehrlich ascites carcinoma (EAC) cells. LHL (250 mg kg-1, i.p.) administered for five consecutive doses at 2-h intervals starting 2 h before DOX (5 mg kg-1, i.p.) single injection, enhanced the antitumour activity of DOX in EAC-bearing mice as manifested by a significant increase in average life span and cure rate of mice. In normal mice, LHL, in the same dose regimen, could not alter the acute cardiotoxicity and lethality of DOX (10 mg kg-1, i.p.). The present data indicate that LHL may improve the therapeutic efficacy of DOX in EAC-bearing mice without compromising its toxicity. Also, our finding supports the LHL/anticancer drug combination approach.

Effects of L-histidine and its structural analogues on human N-myristoyltransferase activity and importance of EEVEH amino acid sequence for enzyme activity.

Myristoyl-CoA:protein N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the cotranslational transfer of myristate to the NH2-terminal glycine residue of a number of important proteins of diverse function. Human NMT (hNMT) activity was found to be activated by L-histidine in a concentration-dependent manner. In contrast, two structural analogues of L-histidine, L-histidinol and histamine, inhibited hNMT activity in a noncompetitive manner with half-maximal inhibitions of 18 and 1.5 mM, respectively. The inhibition of hNMT activity by L-histidinol was reversed by a 2-fold molar excess of L-histidine, suggesting that L-histidine and L-histidinol were competing for a common site on NMT. Kinetic data indicated that whereas L-histidine enhanced the Vmax, both L-histidinol and histamine decreased the Vmax; none of these compounds altered the Km. Our studies suggest that L-histidine and its analogues may be interacting with His-293, involved in myristoyl-CoA transfer, rather than His-218, and implicated in the transfer of myristoyl-CoA to the peptide substrates. Site-directed mutagenesis of His-293, Val-291, and Glu-290 resulted in proteins with no measurable NMT activity. The most conserved region in the catalytic domain EEVEH (289-293) is critical for the myristoyl-CoA transfer in the NMT-catalyzed reactions. This region will be useful for the design of regulators of NMT function.

Effect of L-histidinol on cisplatin nephrotoxicity in the rat.

The effect of L-histidinol (LHL) on the acute nephrotoxicity produced by cisplatin (CDDP; 6 mg/kg, i.v.) was investigated in the rat. Intraperitoneal administration of LHL (100 mg/kg x 5 doses, 2 h apart) starting 2 h prior to CDDP single injection produced significant protection of renal function. The attenuation of nephrotoxicity was evidenced by significant reductions in serum urea and creatinine concentrations, decreased polyuria, reduction in body weight loss, marked reduction in urinary fractional sodium excretion and glutathione-S-transferase (GST) activity, and increased urine/serum creatinine ratio as well as increased creatinine clearance. LHL significantly ameliorated the toxic renal biochemical changes induced by CDDP. Renal lipid peroxides, glutathione levels and GST activity showed a marked tendency towards the normal values. Accumulation of platinum in renal tissues was significantly decreased in the presence of LHL. It is concluded that LHL can act as a nephroprotectant, and it is suggested that it would have beneficial effects on the kidney in clinical settings.

L-histidinol selectively modulates daunomycin toxicity in normal and tumorigenic kidney epithelial cells.

L-Histidinol protects normal cells from anticancer drugs while enhancing the ability of these agents to eradicate tumor cells. We now report that this attribute, previously documented in normal and tumor cells of fibroblastic or myeloid origin, extends to epithelial lines. Clonogenic cell survival assays showed that L-histidinol protected the normal Madin-Darby canine kidney (MDCK) epithelial cell line from daunomycin (DAU) toxicity, but enhanced DAU toxicity in MDCK-T1, a tumorigenic derivative of MDCK. The protection of MDCK cells from DAU by L-histidinol was improved by increasing L-histidine in the media, a condition known to diminish L-histidinol's capacity to inhibit protein synthesis. In contrast, similar conditions markedly diminished the capacity of L-histidinol to enhance DAU killing of MDCK-T1 cells. These data suggest that the differential effects of L-histidinol on DAU toxicity cannot be attributed totally to its ability to inhibit protein synthesis.

Mimetics of L-histidinol which selectively modulate daunomycin toxicity in normal and tumorigenic epithelial cells.

In the accompanying publication, it was shown that L-histidinol protected the normal Madin-Darby canine kidney (MDCK) epithelial cell line from daunomycin (DAU) toxicity, but enhanced DAU toxicity in MDCK-T1, a tumorigenic derivative of MDCK. The protection of MDCK cells from DAU by L-histidinol was also shown to be independent of its ability to inhibit protein synthesis. Here, clonogenic cell survival assays show that imidazole was as effective as L-histidinol in protecting MDCK cells from DAU, but had less impact on MDCK-T1 line. Certain antieicosanoids and antihistamines mimicked, to varying extents, the DAU-modulating action of L-histidinol. These data suggest that the selective modulation of DAU toxicity by L-histidinol involves both inhibition of protein synthesis and unknown action(s) attributable to its imidazole moiety and that other pharmacological agents are modulators of anticancer drug toxicity.

L-histidinol reverses resistance to cisplatinum and other antineoplastics in a tumorigenic epithelial cell line.

L-Histidinol protects normal cells from various anticancer drugs, while enhancing the toxicity of the same agents in drug-sensitive and multidrug-resistant tumor cells. Here, we report that L-histidinol circumvents a novel form of multiple-drug resistance in the MDCK-T1 line, a tumorigenic derivative of the phenotypically-normal Madin-Darby canine kidney (MDCK) epithelial cell line. Clonogenic cells survival assays showed that, compared to the parental MDCK line, the MDCK-T1 line was resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea (approximately 15 fold), to cisplatinum (approximately 10 fold), to 5-fluorouracil (approximately 10-fold) and to cytosine arabinoside (approximately 15-fold). L-Histidinol reversed the resistance of MDCK-T1 cells to these anticancer drugs while protecting the parental MDCK line from these agents. These studies indicate that L-histidinol reverses a unique form of drug-resistance in MDCK-T1 cells by a mechanism dependent upon protein synthesis inhibition.

Cloning of rat asparagine synthetase and specificity of the amino acid-dependent control of its mRNA content.

A full-length cDNA clone for rat asparagine synthetase (AS) was isolated from a cDNA library enriched for amino acid-regulated sequences. The AS cDNA was used to investigate the amino acid-dependent repression of AS mRNA content in rat Fao hepatoma cells. In response to complete amino acid starvation, there was an approximately 10-fold increase in the level of AS mRNA. Three species of mRNA, of approx. sizes 2.0, 2.5 and 4.0 kb, were detected and each was simultaneously regulated to the same degree. The expression of AS mRNA increased by 6 h after removal of amino acids, reached a plateau after 9 h, and was blocked by either actinomycin D or cycloheximide. Partial repression of the AS mRNA content was maintained by the presence of a single amino acid in the culture medium, but the degree of effectiveness for each one varied widely. Glutamine showed the greatest ability to repress the AS mRNA content, even at an extracellular concentration 10 times below its plasma level. Other effective repressors included the amino acids asparagine, histidine and leucine, as well as ammonia. Depletion of selected single amino acids from an otherwise complete culture medium also caused up-regulation. In particular, removal of histidine, threonine or tryptophan from the medium, or the addition of histidinol to inhibit histidinyl-tRNA synthetase, resulted in a significant increase in AS mRNA content. The data indicate that nutrient regulation of AS mRNA occurs by a general control mechanism that is responsive to a spectrum of amino acids.

L-histidinol increases the vulnerability of cultured human leukemia and lymphoma cells to anticancer drugs.

L-Histidinol, a structural analogue of the essential amino acid L-histidine, enhances the toxicity of a variety of anticancer drugs for many tumor cells of animal origin. In this study, the effects of L-histidinol on the proliferation and anticancer drug susceptibility of two human tumor cell lines of lymphoid origin, Daudi and MOLT 4, have been examined. L-Histidinol increased the inherent capacity of six different antineoplastic agents to kill these human tumor cells, in a dose- and time-dependent manner, in spite of the observation that it slowed cell cycle progression in both lines.

Inactivation of histidine ammonia-lyase from Streptomyces griseus by dicarbonyl reagents.

Histidine ammonia-lyase from Streptomyces griseus was inactivated by methylglyoxal and phenylglyoxal, dicarbonyl reagents known to react specifically with arginyl residues in proteins. The inactivation showed pseudo-first-order kinetics and could be prevented by protection with histidinol phosphate, a competitive inhibitor of histidine ammonia-lyase. Analysis of the amino acid composition of histidine ammonia-lyase after treatment with phenylglyoxal, together with the kinetics of inactivation, suggested that inactivation was a consequence of specific reaction with one or more essential arginyl residues at or near the active site of the enzyme.

L-histidinol in experimental cancer chemotherapy: improving the selectivity and efficacy of anticancer drugs, eliminating metastatic disease and reversing the multidrug-resistant phenotype.

Human cancer chemotherapy is limited by two major problems: the failure of commonly used anticancer drugs to act against tumor cells in a specific manner and the ability of malignant cells to resist killing by antineoplastic agents. Experimentally, both of these problems can be solved by using L-histidinol in combination with conventional anticancer drugs. A structural analogue of the essential amino acid L-histidine and an inhibitor of protein biosynthesis. L-histidinol improves the selectivity and the efficacy of a variety of cancer drugs in several transplantable murine tumors. Furthermore, L-histidinol circumvents the drug-resistant traits of a variety of cancer cells, including those showing multidrug resistance. This review will summarize these properties of L-histidinol, present new evidence on its ability to increase the vulnerability of both drug-sensitive and drug-resistant human leukemia cells to various anticancer drugs, and show that, in addition to inhibiting protein synthesis, L-histidinol acts as an intracellular histamine antagonist. The establishment of a connection between the latter mechanism and the capacity to modulate anticancer drug action has resulted in a clinical trial in the treatment of human cancer.

Inhibition of protein synthesis and heat protection: histidinol-resistant mutant cell lines.

The mechanism of histidinol (HST)-induced heat protection was investigated to test the hypothesis that the cessation of protein synthesis itself is one of the events involved in heat protection. For this study, we isolated three HST-resistant mutant strains. HST (5 mM), which inhibited protein synthesis by 88% in the wild type, caused only 0, 9, and 25% inhibition in three mutants, respectively. The drug, which afforded heat protection, (i.e., a 125-fold increase in survival from 4 x 10(-3) to 5 x 10(-1) after 2 hr at 43 degrees C in wild type), did not protect mutant cells from heat killing. In contrast, cycloheximide (10 micrograms/ml) which inhibited protein synthesis by 95% in both wild type and mutant cell types, protected both cell types from heat killing. Therefore, these results suggest that the cessation of protein synthesis, per se, preventing synthesis of nascent polypeptides, is a major event leading to heat protection.

Increased therapeutic index of antineoplastic drugs in combination with intracellular histamine antagonists.

L-Histidinol, a protein synthesis inhibitor and structural analogue of L-histidine, has been demonstrated in chemotherapy-treated mice to be cytoprotective to normal stem cells but to enhance cytotoxicity to tumor cells. N,N-Diethyl-2-[4-(phenylmethyl) phenoxy]ethanamine.HCl (DPPE) is an antagonist of recently described microsomal and nuclear intracellular histamine receptors implicated in the mediation of proliferation and modulation of prostaglandin synthesis. DPPE is cytotoxic to tumor cells in vitro and cytoprotective to the gut in vivo. Noting the similar pharmacologic profiles for histidinol and DPPE and the structural resemblance between histidinol and histamine, we tested 1) whether binding to intracellular histamine receptors may be important to the action of histidinol, 2) whether there exists a differential effect of DPPE and histidinol on proliferating normal and transformed or malignant cells, and 3) whether DPPE, like histidinol, protects host cells from the effects of chemotherapy while augmenting tumor cell kill in vivo. It was observed that histidinol does compete at intracellular histamine receptors in isolated microsomes and nuclei, but with significantly lower affinity than DPPE. Nevertheless, for each agent, potency at intracellular histamine receptors correlates with potency to inhibit DNA and protein synthesis, without cytotoxicity, in normal mitogen-stimulated murine lymphocytes and to kill transformed mouse lymphocytes or MCF-7 human breast cancer cells. As demonstrated previously for histidinol (1-2 g/kg), DPPE (4 mg/kg) protected murine bone marrow progenitors from doxorubicin or fluorouracil, while doses of 4-50 mg/kg significantly enhanced the antitumor activity of doxorubicin and daunorubicin in murine models of early cancer. One postulate to explain the effects of intracellular histamine receptor ligands is that intracellular histamine mediates DNA and protein synthesis, possibly through a downward modulation of growth-inhibitory prostaglandin levels. Antagonism of the intracellular action of histamine at intracellular histamine receptors by DPPE or histidinol may result in differential perturbations of growth/eicosanoid metabolism in normal and malignant cells, thus forming the basis of a new approach to chemotherapy.