Choline kinase alpha-Putting the ChoK-hold on tumor metabolism.

It is well established that lipid metabolism is drastically altered during tumor development and response to therapy. Choline kinase alpha (ChoKα) is a key mediator of these changes, as it represents the first committed step in the Kennedy pathway of phosphatidylcholine biosynthesis and ChoKα expression is upregulated in many human cancers. ChoKα activity is associated with drug resistant, metastatic, and malignant phenotypes, and represents a robust biomarker and therapeutic target in cancer. Effective ChoKα inhibitors have been developed and have recently entered clinical trials. ChoKα's clinical relevance was, until recently, attributed solely to its production of second messenger intermediates of phospholipid synthesis. The recent discovery of a non-catalytic scaffolding function of ChoKα may link growth receptor signaling to lipid biogenesis and requires a reinterpretation of the design and validation of ChoKα inhibitors. Advances in positron emission tomography, magnetic resonance spectroscopy, and optical imaging methods now allow for a comprehensive understanding of ChoKα expression and activity in vivo. We will review the current understanding of ChoKα metabolism, its role in tumor biology and the development and validation of targeted therapies and companion diagnostics for this important regulatory enzyme. This comes at a critical time as ChoKα-targeting programs receive more clinical interest.

Crystal structures of human choline kinase isoforms in complex with hemicholinium-3: single amino acid near the active site influences inhibitor sensitivity.

Human choline kinase (ChoK) catalyzes the first reaction in phosphatidylcholine biosynthesis and exists as ChoKalpha (alpha1 and alpha2) and ChoKbeta isoforms. Recent studies suggest that ChoK is implicated in tumorigenesis and emerging as an attractive target for anticancer chemotherapy. To extend our understanding of the molecular mechanism of ChoK inhibition, we have determined the high resolution x-ray structures of the ChoKalpha1 and ChoKbeta isoforms in complex with hemicholinium-3 (HC-3), a known inhibitor of ChoK. In both structures, HC-3 bound at the conserved hydrophobic groove on the C-terminal lobe. One of the HC-3 oxazinium rings complexed with ChoKalpha1 occupied the choline-binding pocket, providing a structural explanation for its inhibitory action. Interestingly, the HC-3 molecule co-crystallized with ChoKbeta was phosphorylated in the choline binding site. This phosphorylation, albeit occurring at a very slow rate, was confirmed experimentally by mass spectroscopy and radioactive assays. Detailed kinetic studies revealed that HC-3 is a much more potent inhibitor for ChoKalpha isoforms (alpha1 and alpha2) compared with ChoKbeta. Mutational studies based on the structures of both inhibitor-bound ChoK complexes demonstrated that Leu-401 of ChoKalpha2 (equivalent to Leu-419 of ChoKalpha1), or the corresponding residue Phe-352 of ChoKbeta, which is one of the hydrophobic residues neighboring the active site, influences the plasticity of the HC-3-binding groove, thereby playing a key role in HC-3 sensitivity and phosphorylation.

Additive and synergistic effects of fetal nicotine and dexamethasone exposure on cholinergic synaptic function in adolescence and adulthood: Implications for the adverse consequences of maternal smoking and pharmacotherapy of preterm delivery.

Maternal smoking contributes to preterm delivery; glucocorticoids are the consensus treatment for prematurity, thus producing fetal coexposure to nicotine and dexamethasone. We administered nicotine to pregnant rats throughout gestation at a dose (3 mg/kg/day) producing plasma levels typical of smokers. Later in gestation, animals received dexamethasone (0.2 mg/kg). We assessed developmental indices for acetylcholine (ACh) synaptic function throughout adolescence, young adulthood and later adulthood, evaluating brain regions possessing major ACh projections and cell bodies; we measured choline acetyltransferase activity, hemicholinium-3 binding to the presynaptic choline transporter and nicotinic ACh receptor binding. In general, nicotine and dexamethasone, alone or in combination, produced regionally-selective increases or decreases in choline acetyltransferase activity but larger, consistent elevations in hemicholinium-3 and nicotinic ACh receptor binding; the patterns were indicative of ACh synaptic hyperactivity. Superimposed on these overall effects, there were significant disparities in temporal and regional relationships among the different treatments, notably involving effects that emerged later in life, after a period of apparent normality. This indicates that nicotine and dexamethasone do not simply produce an initial ACh neuronal injury that then persists throughout the lifespan but rather, they alter the developmental trajectory of ACh function. Most importantly, the combined exposure to nicotine + dexamethasone elicited greater changes than either of the individual exposures, involving both additive and synergistic effects. Our results thus point to potentially worse neurobehavioral outcomes of the pharmacotherapy of preterm labor in the offspring of smokers.

A novel choline cotransporter sequestration compartment in cholinergic neurons revealed by selective endosomal ablation.

The sodium-dependent, high affinity choline transporter - choline cotransporter - (ChCoT, aka: cho-1, CHT1, CHT) undergoes constitutive and regulated trafficking between the plasma membrane and cytoplasmic compartments. The pathways and regulatory mechanisms of this trafficking are not well understood. We report herein studies involving selective endosomal ablation to further our understanding of the trafficking of the ChCoT. Selective ablation of early sorting and recycling endosomes resulted in a decrease of approximately 75% of [3H]choline uptake and approximately 70% of [3H]hemicholinium-3 binding. Western blot analysis showed that ablation produced a similar decrease in ChCoTs in the plasma membrane subcellular fraction. The time frame for this loss was approximately 2 h which has been shown to be the constitutive cycling time for ChCoTs in this tissue. Ablation appears to be dependent on the intracellular cycling of transferrin-conjugated horseradish peroxidase and the selective deposition of transferrin-conjugated horseradish peroxidase in early endosomes, both sorting and recycling. Ablated brain slices retained their capacity to recruit via regulated trafficking ChCoTs to the plasma membrane. This recruitment of ChCoTs suggests that the recruitable compartment is distinct from the early endosomes. It will be necessary to do further studies to identify the novel sequestration compartment supportive of the ChCoT regulated trafficking.

Activity and subcellular trafficking of the sodium-coupled choline transporter CHT is regulated acutely by peroxynitrite.

Excess formation of nitric oxide and superoxide by-products (peroxynitrite, reactive oxygen, and reactive nitrogen species) attenuates cholinergic transmission potentially having a role in Alzheimer disease pathogenesis. In this study, we investigated mechanisms by which acute exposure to peroxynitrite impairs function of the sodium-dependent hemicholinium-3 (HC-3)-sensitive choline transporter (CHT) that provides substrate for acetylcholine synthesis. The peroxynitrite generator 3-morpholinosydnonimine (SIN-1) acutely inhibited choline uptake in cells stably expressing FLAG-tagged rat CHT in a dose- and time-dependent manner, with an IC(50) = 0.9 +/- 0.14 mM and t((1/2)) = 4 min. SIN-1 significantly reduced V(max) of choline uptake without altering the K(m). This correlated with a SIN-1-induced decrease in cell surface CHT protein, observed as lowered levels of HC-3 binding and biotinylated CHT at the plasma membrane. It is noteworthy that short-term exposure of cells to SIN-1 accelerated the rate of internalization of CHT from the plasma membrane, but it did not alter return of CHT back to the cell surface. SIN-1 did not disrupt cell membrane integrity or cause cell death. Thus, the inhibitory effect of SIN-1 on choline uptake activity and HC-3 binding was related to enhanced internalization of CHT proteins from the plasma membrane to subcellular organelles.

Altered localization of choline transporter sites in the mouse hippocampus after prenatal heroin exposure.

Prenatal heroin exposure disrupts hippocampal cholinergic synaptic function and related behaviors. Biochemical studies indicate an increase in the number of presynaptic high-affinity choline transporter (HACT) sites, as assessed by [3H]hemicholinium-3 (HC-3) binding. The present study was designed to assess whether this effect involves global upregulation of the transporter, or whether disruption occurs with a specific tempero-spatial distribution. Pregnant mice were given 10mg/kg per day of heroin subcutaneously on gestational days (GD) 9-18. Autoradiographic distribution of HC-3 binding sites was evaluated in the hippocampus of the offspring at postnatal days 15, 25, and 53. These results, suggestive of hippocampal "miswiring," are likely to explain the net impairment of cholinergic synaptic function after prenatal heroin exposure, despite the simultaneous upregulation of both presynaptic cholinergic activity and postsynaptic receptors. Understanding the subregional selectivity of hippocampal defects can lead to the development of strategies that may potentially enable therapeutic interventions to offset or reverse the neurobehavioral defects.

Par-4 inhibits choline uptake by interacting with CHT1 and reducing its incorporation on the plasma membrane.

CHT1 is a Na(+)- and Cl(-)-dependent, hemicholinium-3 (HC-3)-sensitive, high affinity choline transporter. Par-4 (prostate apoptosis response-4) is a leucine zipper protein involved in neuronal degeneration and cholinergic signaling in Alzheimer's disease. We now report that Par-4 is a negative regulator of CHT1 choline uptake activity. Transfection of neural IMR-32 cells with human CHT1 conferred Na(+)-dependent, HC-3-sensitive choline uptake that was effectively inhibited by cotransfection of Par-4. Mapping studies indicated that the C-terminal half of Par-4 was physically involved in interacting with CHT1, and the absence of Par-4.CHT1 complex formation precluded the loss of CHT1-mediated choline uptake induced by Par-4, indicating that Par-4.CHT1 complex formation is essential. Kinetic and cell-surface biotinylation assays showed that Par-4 inhibited CHT1-mediated choline uptake by reducing CHT1 expression in the plasma membrane without significantly altering the affinity of CHT1 for choline or HC-3. These results suggest that Par-4 is directly involved in regulating choline uptake by interacting with CHT1 and by reducing its incorporation on the cell surface.

Effects of perinatal nicotine exposure on development of [3H]hemicholinium-3 binding sites in rat neonate brain.

In this study, [3H]hemicholinium-3 ([3H]HC-3) binding, which labels the presynaptic high affinity-choline transport sites, was examined in two brain regions, cerebral cortex and midbrain, of nicotine-treated and -untreated rat neonates. In nicotine-untreated neonates, [3H]HC-3 binding sites of cerebral cortex increased from 64 fmol/mg protein at postnatal day 7 to 142 fmol/mg protein at postnatal day 35. In nicotine-treated neonates, the development of [3H]HC-3 binding sites in cerebral cortex was significantly retarded, compared with control neonates on the 7th, 14th and 21st postnatal days. In parallel with this, the development of muscarinic receptor in cerebral cortex, which was detected by [3H]quinuclidinyl benzylate ([3H]QNB) binding, was also retarded by nicotine treatment. However, in midbrain, neither [3H]HC-3 nor [3H]QNB binding sites at postnatal day 14 was affected by nicotine treatment. These results strongly suggest that perinatal treatment with nicotine inhibits presynaptic and postsynaptic development of the cholinergic pathway in cerebral cortex but not in midbrain of rat neonate.

Modeling adolescent nicotine exposure: effects on cholinergic systems in rat brain regions.

Smoking among teenagers is increasing and the initiation of tobacco use during adolescence is associated with subsequently higher cigarette consumption and lower rates of quitting. Few animal studies have addressed whether adolescent nicotine exposure exerts unique or lasting effects on brain structure or function. Initial investigations with a rat model of adolescent nicotine exposure have demonstrated that the vulnerable developmental period for nicotine-induced brain cell damage extends into adolescence. In the current study, we examined the effect of nicotine on cholinergic systems in male and female adolescent rats with an infusion paradigm designed to match the plasma levels found in human smokers or in users of the transdermal nicotine patch. Choline acetyltransferase activity (ChAT) and [3H]hemicholinium-3 binding (HC-3) were monitored; ChAT is a static marker that closely reflects the density of cholinergic innervation, whereas HC-3 binding, which labels the presynaptic high-affinity choline transporter, is responsive additionally to nerve impulse activity. Measurements were carried out in the midbrain, the region most closely involved in reward and addiction pathways, as well as in the cerebral cortex and hippocampus. During nicotine treatment and for 1 month after the termination of treatment, ChAT activity was reduced significantly in the midbrain but not in the other regions. HC-3 binding showed a substantial increase during the course of nicotine treatment and again, the effect was limited to the midbrain. Midbrain values returned to normal immediately after the cessation of nicotine exposure and then showed a subsequent, transient suppression of activity. Although the cerebral cortex showed little or no change in HC-3 binding during or after nicotine administration, activity was reduced persistently in the hippocampus. The regionally-selective effects of adolescent nicotine treatment on cholinergic systems support the concept that adolescence is a vulnerable developmental period for ultimate effects on behavior.

Early increase in choline kinase activity upon induction of the H-ras oncogene in mouse fibroblast cell lines.

The effects of expression of the H-ras oncogene on phosphatidylcholine metabolism were examined in C3H10T1/2 and NIH3T3 cells expressing ras constitutively or under the control of inducible promoters. Cell lines expressing ras under the control of the mouse metallothionein promoter and the Escherichia coli lac operator/repressor system and an NIH3T3 cell line stably transfected with the ras oncogene were studied. Phosphocholine levels were elevated in the cells constitutively expressing ras and were increased 4-6 h upon induction in the inducible cell lines. Glycerophosphocholine, which is elevated five- to sixfold in constitutively transfected ras cells, did not increase at early times of induction, suggesting the absence of increased phosphatidylcholine degradation via a phospholipase A. Choline kinase activity increased within 4-6 h upon induction and correlated well with the increase in phosphocholine levels. This increase in phosphocholine levels could be prevented by the addition of hemicholinium-3, a competitive inhibitor of choline kinase. Expression of activated c-raf or v-raf also increased choline kinase activity, suggesting that the induction of choline kinase by ras is downstream of the ras/raf interaction. Long-term and short-term labeling experiments failed to detect evidence for increased phospholipase C activity. These results suggest that the increase in choline kinase activity observed in cells expressing ras is an early, integral part of ras transformation and is the main contributor to increased phosphocholine levels accompanying morphological changes.

Transport of choline by Madin-Darby canine kidney cells.

Choline is an essential precursor for the synthesis of phosphatidylcholine, the most abundant phospholipid classes in renal cells, as well as for the synthesis of the osmolyte glycerophosphorylcholine. The characteristics of choline uptake in the renal epithelial cell line MDCK were investigated. In the range of physiological concentrations, choline entered MDCK cells, grown as a monolayer on solid support, via a specific sodium-independent transport system (apparent Km = 43 microM, apparent Vmax = 284 pmol/mg protein per 5 min). Cell ATP depletion, addition of KCl to the medium to reduce the cell membrane potential, and hemicholinium-3 (HC-3) inhibited choline uptake. Specific binding of [3H]HC-3 was detected on the apical membrane of cells grown on plastic dishes, whereas it occurred only on the basolateral domain of cells grown on permeant support. When growing cells on filter, choline uptake from the basolateral side was 10-times the apical uptake. This suggests that the choline carrier present at the apical domain of cells grown on solid support is either inactivated or no longer targeted to the apical but to the basolateral membrane of MDCK cells grown on filter.

Effects of NBM lesions with two neurotoxins on spatial memory and autoshaping.

Four groups of Wistar rats received either vehicle, quisqualate, or one of two different ibotenic acid infusions into the basal forebrain. Following recovery from surgery, all rats were tested in three distinct behavioral paradigms: the Bättig radial arm maze, the Barnes circular platform, and autoshaping in an operant chamber. The results showed that the size and site of the ibotenic acid lesion had a profound effect on acquisition performance in some, but not all, procedures. Performance in the Bättig maze and acquisition of a food-rewarded lever press were in particular disrupted by ibotenic acid lesions. The severity of the reduction in cortical choline acetyltransferase (ChAT) did not correlate with performance in the tests. Quisqualate produced the largest reduction in ChAT levels but had no significant effect on performance in any of the three procedures used. Anatomic analysis revealed severe nonspecific damage to the striatum following ibotenic acid that was more pronounced in the group receiving a highly concentrated solution of ibotenic acid as compared to rats infused with a greater volume but less concentrated solution of the neurotoxin. Striatal damage was much less severe following quisqualic acid infusions. However, both types of neurotoxins produced equivalent nonspecific degeneration of the reticular thalamic nucleus. These data confirm reports that nonspecific damage appears to define the severity of ibotenic acid lesions on subsequent behavioral performance.

Deficits in development of central cholinergic pathways caused by fetal nicotine exposure: differential effects on choline acetyltransferase activity and [3H]hemicholinium-3 binding.

Nicotine has been hypothesized to induce neurobehavioral teratology by mimicking prematurely the natural developmental signals ordinarily communicated by the ontogeny of cholinergic synaptic transmission. In the current study, the effects of fetal nicotine exposure (2 mg/kg/day or 6 mg/kg/day) on development of central cholinergic pathways were examined in striatum and hippocampus of animals exposed from gestational days 4 through 20, using maternal infusions with osmotic minipumps. Brain region weights and choline acetyltransferase activity, an enzymatic marker for development of cholinergic nerve terminals, were within normal limits in the nicotine-exposed animals. However, development of [3H]hemicholinium-3 binding which labels the presynaptic high affinity cholinergic transporter, was deficient in both striatum and hippocampus. Abnormalities occurred during two distinct phases; in the early neonatal period, when [3H]hemicholinium-3 binding sites are transiently overexpressed, and during or after the period of rapid synaptogenesis, when binding in controls is rising consequent to the increase in nerve impulse activity. These data thus indicate that fetal nicotine exposure, even at doses that do not cause overt signs of maternal/fetal/neonatal toxicity or growth impairment, influences both specific gene expression of cholinergic nerve terminal markers, as well as indices of neuronal function. Comparison of regional selectivity at the two dose levels indicated greater sensitivity of the striatum, a region with a prenatal peak of neuronal mitosis, as compared to hippocampus, where mitosis peaks postnatally; the regional differences are consistent with vulnerability to nicotine during a critical phase of cell development.

High-affinity choline transport regulation by drug administration during postnatal development.

High-affinity choline transport sites specifically bind [3H]hemicholinium-3. Hemicholinium-3 binding sites are regulated by in vivo drug treatments in the same manner as these drugs alter acetylcholine release and high-affinity choline transport. The current study examines regulation of binding sites by in vivo drug administration for adult, day 15, and day 5 rats. Drugs or saline were administered intraperitoneally, and striatal and cortical membrane preparations were assayed. Control [3H]hemicholinium-3 binding increases twofold between postnatal days 5 and 15 only in striatum. After day 15, binding increases 2.7-fold in cortex and striatum. Nicotine treatment increases striatal and cortical hemicholinium-3 binding at all three ages, with greater percent increases at day 5. Haloperidol increases binding only in striatum, again with larger effects at day 5. Both striatal and cortical binding are reduced by oxotremorine; however, the magnitude of this effect is unchanged during development. Pentobarbital reduces binding only in striatum, with no developmental change. Atropine and apomorphine do not change binding from control values. In summary, all drug treatments effective in adults were already effective by day 5. Cholinergic terminals present early in development are regulated by similar nicotinic and muscarinic cholinergic, dopaminergic, and sedative-hypnotic mechanisms as the adult. Changes in magnitude may be due to changes in drug metabolism or to developmental differences in regulation.

Ca2(+)-dependent, ATP-induced conversion of the [3H]hemicholinium-3 binding sites from high- to low-affinity states in rat striatum: effect of protein kinase inhibitors on this affinity conversion and synaptosomal choline transport.

Tritium-labeled hemicholinium-3 ([3H]HC-3) was used to characterize the sodium-dependent high-affinity choline carrier sites in rat striatal preparations. In an earlier study, we had shown that [3H]HC-3 labels choline carrier sites with high and low affinities and had suggested that the low-affinity sites represent "functional" carrier sites. The objective of the present study was to examine the mechanisms involved in the regulation of the two affinity states of [3H]HC-3 binding. Here, we demonstrate that these two affinity states are totally interconvertible; addition of 0.1 mM ATP in the binding assay medium quantitatively converted all the binding sites to the low-affinity state, whereas addition of 1 mM beta,gamma-methylene 5'-ATP quantitatively converted all the binding sites to the high-affinity state. Preincubation of the tissue (for 15 min at 37 degrees C) before the binding assay also converted the binding sites to the high-affinity state, whereas supplementation of the assay medium with ATP (0.5 mM) again induced expression of the low-affinity state of the binding sites. This effect of ATP was found to be selective for this nucleotide. Neither ADP (1 mM) nor cyclic AMP could mimic such an effect. Other nucleotide triphosphates--CTP (0.5 mM) and GTP (0.5 mM)--also could not substitute for ATP. GTP, however, caused nearly a 35% reduction in the number of binding sites, accompanying a loss of the low-affinity component of binding. This effect of GTP was also shared by 5'-guanylylimidodiphosphate but not by GDP or cyclic GMP. This ATP-dependent low-affinity conversion of [3H]HC-3 binding sites requires divalent metal ions.(ABSTRACT TRUNCATED AT 250 WORDS)

Specificity of the activation of [3H]hemicholinium-3 binding by phospholipase A2.

Phospholipase A2 (PLA2) treatment has been shown previously to stimulate the sodium-dependent high-affinity choline uptake system as assessed by both the specific binding of [3H]hemicholinium-3 ([ 3H]HCh-3) and the uptake of [3H]choline. In the present study, the specificity of PLA2-induced stimulation upon [3H]HCh-3 binding has been examined. PLA2, as well as phospholipase C (PLC), treatment of synaptic membranes produced a dose-dependent increase in the specific binding of [3H]HCh-3 whereas neither phospholipase B nor phospholipase D had any effect. PLC-induced stimulation of [3H]HCh-3 binding resulted from a significant decrease in the Kd without a change in the maximum binding of [3H]HCh-3 binding. PLC treatment of synaptosomes resulted in an inhibition of [3H]choline uptake accompanied by an inhibition of Na+, K+-adenosine triphosphatase activity. In contrast to the increase of [3H]HCh-3 binding, the specific binding of both [3H]desipramine and [3H]mazindol was decreased by PLA2 treatment. After PLA2 treatment, [3H]HCh-3 binding was increased about 2.5-fold over basal levels in different regions of the brain. Electrolytic lesions of the medial septal nucleus and kainic acid-induced lesions of the striatum resulted in a marked reduction of [3H]HCh-3 binding in the hippocampus and the striatum, respectively. Residual [3H]HCh-3 binding in the denervated hippocampus and lesioned striatum was increased by PLA2 treatment but remained lower than that in PLA2-treated controls. Finally, atropine-induced up-regulation of [3H]HCh-3 binding in vivo was not additive with PLA2-induced stimulation. These results support the hypothesis that PLA2 might be involved in the regulation of the sodium-dependent high-affinity choline uptake.