Nuclear localization of soluble CTP:phosphocholine cytidylyltransferase

The soluble form of CTP:phosphocholine cytidylyltransferase, which has previously been assumed to be cytosolic, has been localized to the nucleus of several cell types. Indirect immunofluorescence microscopy indicated a nuclear location in HepG2, NIH-3T3, and L-cells. A comparison of the fluorescence pattern of wild-type CHO cells with a cytidylyltransferase-deficient mutant provided genetic evidence that cytidylyltransferase is nuclear in CHO cells. The enzyme is also predominantly nuclear in rat liver, as revealed by staining frozen sections of that tissue. When L-cells were fractionated by enucleation, over 95% of cytidylyltransferase activity was found in the nuclear fraction, providing biochemical evidence for a nuclear location in these cells. In light of the demonstration that the membrane-bound cytidylyltransferase in CHO cells is associated with the nuclear envelope (Watkins, J. D., and Kent, C. (1992) J. Biol. Chem. 267, 5686-5692), these results suggest that this enzyme is predominantly an intranuclear enzyme.     

Activation of CTP:phosphocholine cytidylyltransferase by hypochlorite-oxidized phosphatidylcholines

The acute psychomotor response and development of sensitization to amphetamine is attenuated if i.p. injections are given in the cage where a rat lives relative to when injections are given in a novel but physically identical test environment. Furthermore, when the environmental cues predicting i.p. injections are completely eliminated by using remotely activated i.v. injections in the home cage, 1.0 mg/kg amphetamine produces a very small acute response and no sensitization. The same treatments do produce sensitization if i.v. injections are signaled by placement of the rat in a novel test cage. The present experiment was designed to determine if there is a similar effect of environmental condition on the response to i.v. cocaine, and to what extent the effect may be dose-dependent. This was accomplished by comparing the psychomotor activating effects (rotational behavior) of repeated i.v. administrations of one of eight doses of cocaine (0.0, 0.3, 0.6, 1.2, 2.4, 3.6, 4.8, or 7.2 mg/kg) given in the home cage, with infusions of the same doses given in a novel test cage. There was no effect of environment on the acute psychomotor response to cocaine. There was, however, a significant effect of environment on the induction of sensitization. A higher dose of cocaine was required to induce sensitization when i.v. administrations were given in the home cage than when they were given in a physically identical but novel test environment. At high doses, however, cocaine induced sensitization regardless of environmental condition. The results suggest that the effect of this environmental manipulation is to shift the dose-effect curve for the induction of sensitization, and support the notion that the ability of psychostimulant drugs to induce sensitization can be modulated by the circumstances surrounding drug administration.     

Identification of an inhibitory domain of CTP:phosphocholine cytidylyltransferase

The function of the putative amphipathic helices between residues 236 and 314 of CTP:phosphocholine cytidylyltransferase was examined by constructing two truncation mutants; CT314 was missing the entire phosphorylation segment, whereas CT236 was missing both the region with the putative amphipathic helices and the phosphorylation segment. Stable cells lines expressing these truncation mutants in Chinese hamster ovary 58 cells were isolated and characterized. CT314 was predominantly soluble in control cells but became membrane-associated in cells treated with oleate, which also causes translocation of wild-type cytidylyltransferase. CT236 was found to be soluble both in control cells and in cells treated to cause translocation. These results strongly suggest that the membrane-binding site is located within residues 237-314. When assayed for activity in vitro, the mutant forms were catalytically active in the presence of exogenous lipids. CT236, moreover, was as active in the absence of lipids as in their presence, whereas CT314 required lipids for activity. The rate of phosphatidylcholine synthesis in cells expressing CT236 was considerably higher than in wild-type cells, consistent with the enzyme being constitutively active in the cells. These results indicate that residues 237-314 constitute an inhibitory segment; when this segment is removed from the catalytic domain by truncation or by binding to membranes, an inhibitory constraint is removed and cytidylyltransferase is activated.     

Immunological characterization, lipid dependence, and subcellular localization of CTP:phosphoethanolamine cytidylyltransferase purified from rat liver. Comparison with CTP:phosphocholine cytidylyltransferase

CTP:phosphoethanolamine cytidylyltransferase (ET) (ethanolamine-phosphate cytidylyltransferase, EC 2.7.7.14), which is generally considered as the rate-regulatory enzyme of phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway, was purified to homogeneity from a rat liver postmicrosomal supernatant. A polyclonal antibody was raised against the enzyme in rabbits and subsequently purified by affinity chromatography. The affinity-purified antibody recognized one single immunoreactive 49.6-kDa protein band on SDS-polyacrylamide gel. The enzyme showed an isoelectric point at a pH of 6.5 and was sensitive to various sulfhydryl reagents. Cross-reactivity experiments of ET and CTP:phosphocholine cytidylyltransferase (CT) (choline-phosphate cytidylyltransferase, EC 2.7.7.15) with their corresponding antibodies showed that these enzymes were immunologically distinct. In contrast with the well known lipid dependence of CT, the activities of both purified and cytosolic ETs were not affected by the presence of various phospholipid preparations. Differential centrifugation studies as well as release experiments with digitonin-permeabilized hepatocytes demonstrated that ET, unlike CT, is not associated with cellular organelles. However, amino acid analysis of ET revealed a high content of hydrophobic amino acids, suggesting a possible association of this enzyme with some kind of cellular structure in the hepatocyte.     

Expression of wild-type and mutant rat liver CTP: phosphocholine cytidylyltransferase in a cytidylyltransferase-deficient Chinese hamster ovary cell line

The strain 58 Chinese hamster ovary (CHO) mutant defective in CTP:phosphocholine cytidylyltransferase was characterized as an expression system for exogenous cytidylyltransferase. Strain 58 cells express less than 5% of the wild-type level of cytidylyltransferase protein at the permissive temperature even though the steady-state messenger RNA levels were found to be similar to those in the parental CHO-K1 cell line. A point mutation from arginine to histidine at amino acid 140 was identified in the strain 58 protein. Rat liver cytidylyltransferase was stably expressed in strain 58 cells and shown to be active, targeted to the nucleus, phosphorylated, and activated by methylethanolamine supplementation or phospholipase C treatment. Thus, the mechanisms by which cytidylyltransferase is processed and regulated in CHO-K1 cells are intact in strain 58 cells. The heterologously expressed protein complemented the strain 58 defects in both temperature-sensitive growth and phosphatidylcholine biosynthesis, consistent with a single lesion in the structural gene for cytidylyltransferase being responsible for both phenomena. Overexpression of cytidylyltransferase activity at levels up to eightfold higher than those in CHO-K1 cells did not appreciably affect phosphatidylcholine metabolism. A putative casein kinase II phosphorylation site was altered by site-directed mutagenesis and expressed in the strain 58 cells. Alteration of this site did not affect expression and regulation of cytidylyltransferase activity.     

Evidence for phosphorylation of CTP:phosphocholine cytidylyltransferase by multiple proline-directed protein kinases

Reversible phosphorylation of CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme of phosphatidylcholine biosynthesis, is thought to play a role in regulating its activity. In the present study, the hypothesis that proline-directed kinases play a major role in phosphorylating cytidylyltransferase is substantiated using a c-Ha-ras-transfected clone of the human keratinocyte cell line HaCaT. Cellular extracts from epidermal growth factor-stimulated HaCaT cells and from ras-transfected HaCaT cells phosphorylated cytidylyltransferase much stronger as compared with extracts from quiescent HaCaT cells. The tryptic phosphopeptide pattern of cytidylyltransferase phosphorylated by cell-free extracts from ras-transfected HaCaT cells was similar compared with the patterns of cytidylyltransferase phosphorylated by p44mpkmitogen-activated protein kinase and p34cdc2 kinase in vitro, whereas in the case of casein kinase II the pattern was different. Furthermore, in c-Ha-ras-transfected HaCaT cells the in vivo phosphorylation state of cytidylyltransferase was 2-fold higher as compared with untransfected HaCaT cells. This higher phosphorylation of cytidylyltransferase in the ras-transfected clone was reduced to a level below the phosphorylation of cytidylyltransferase in untransfected cells, using olomoucine, a specific inhibitor of proline-directed kinases. The reduced phosphorylation of cytidylyltransferase in olomoucine-treated cells correlated with an enhanced stimulation of enzyme activity by oleic acid.     

Lipid activation of CTP:phosphocholine cytidylyltransferase is regulated by the phosphorylated carboxyl-terminal domain

The role of the phosphorylated carboxyl-terminal domain of CTP:phosphocholine cytidylyltransferase (CT) in the regulation of enzyme activity was investigated by comparing the catalytic properties of wild-type CT to two mutant proteins with altered carboxyl-terminal phosphorylation domains. CT isolated from a baculovirus expression system was extensively phosphorylated at multiple sites in the carboxyl-terminal domain. The CT[S315A] mutant lacked a major CT phosphorylation site, and the carboxyl-terminal deletion mutant, CT[delta 312-367], was not phosphorylated. The higher activities of CT[delta 312-367] and CT[S315A] relative to CT were attributed to differences in the sensitivities of the enzymes to lipid activators. The rank order of the apparent Km values for activation by either phosphatidylcholine/oleic acid or phosphatidylcholine/diacylglycerol was CT > CT[S315A] > CT[delta 312-367]. In addition, CT exhibited negative cooperativity in its activation by phosphatidylcholine/oleic acid (nH = 0.64) and phosphatidylcholine/diacylglycerol (nH = 0.74) vesicles, whereas CT[delta 312-367] and CT[S315A] did not. These data support the concept that the phosphorylation of the CT carboxyl-terminal domain interferes with the activation of CT by lipid regulators.     

Identification of a putative membrane-interacting domain of CTP:phosphocholine cytidylyltransferase from rat liver

A putative membrane-interacting domain of CTP:phosphocholine cytidylyltransferase (CT) was identified using two peptide-specific antibodies. One antibody (SA2) was raised against the N-terminus of CT (amino acid residues 1-17) and the other antibody (SA209) against an alpha-helical domain of the enzyme (amino acid residues 247-257). Both antibodies quantitatively immunoprecipitated CT from rat liver cytosol and showed specificity towards CT when octylglucoside extracts of rat liver cytosol were assessed by Western blot analysis. However, further experiments revealed that the antibodies had different characteristics. Whereas the antibody directed against the N-terminus of CT (SA2) did not influence CT/membrane interaction, the new antibody (SA209) against the alpha-helical domain of the enzyme interfered with this interaction. Our results provide experimental evidence that the alpha-helical domain (amino acid residues 228-287) of CT may serve as a membrane-interacting domain.     

The direct effect of anticancer agents on the telomerase activity in human cancer cell lines

Telomerase, a ribonucleoprotein that maintains the ends of linear eukaryotic chromosomes, is expressed in the majority of malignant cells, while most normal somatic cells have no telomerase activity except germline and stem cells. Therefore, telomerase activity is considered one of important characteristics of tumors. In reviewing the possibility that conventional anticancer agents can partly perform their functions through modulation of telomerase. Several data including ours suggest that the down-modulation of telomerase activity along with inhibition by agents is the secondary event associated with cell death. Moreover, cells growth arrested at specific phase of cell cycle by agents still show the high level of telomerase activity. Since telomerase activity well correlates to the cell viability of treated cells, to study telomerase provides us a new implement to examine the sensitivity of cancer cells to agents.     

Alkylphosphocholines: Effects on human leukemic cell lines and normal bone marrow cells

The anti-leukemic activity of a series of alkylphosphocholines (APCs) was studied against a panel of human leukemic cell lines (HL-60, K-562, Reh, MOLT-4, Jurkat, Ramos and Raji). Cytotoxic efficacy was measured by the MTT cell survival assay. All cell lines were found to be sensitive, except the multipotential CML-derived K-562 cell line. Flow cytometry of HL-60 cells showed a significant decrease of cells in S phase and the formation of a sub-G fraction. DNA fragmentation typical for programmed cell death was detected by DNA gel electrophoresis in these cells but not in any of the other leukemic lines. At concentrations below the cytotoxic range, mitogenic effects were seen in HL-60 cells after 14-hr exposure. Colony formation by K-562 cells revealed an augmented clonogenicity after exposure to APC with a short alkyl chain. In contrast, cells of lymphoid origin did not undergo DNA fragmentation or show mitogenic stimulation after exposure to APC. Normal bone marrow cells were also investigated for mitogenic and genotoxic effects. No decrease was found in the number of hematopoietic progenitors in long-term bone marrow cell cultures after exposure to APC. On the contrary, a significant increase was found after short exposure. Dodecylphosphocholine, hexadecylphosphocholine (HPC) and (octadecyl-[2-(N-methylpiperidino)-ethyl]phosphate exhibited a mild clastogenicity at equimolar high doses on murine bone marrow cells in vivo, which is unusual for the majority of classical DNA-interacting anti-cancer drugs. In conclusion, APCs are agents with a broad spectrum of in vitro anti-leukemic effects, which lack hematological toxicity.     

Inhibitors of tyrosine phosphorylation induce apoptosis in human leukemic cell lines

Experimental evidence suggests that hematopoietic growth factors promote cell survival by suppressing apoptosis or programmed cell death. Since interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) induce tyrosine phosphorylation of a common set of proteins in the factor-dependent cell line M07e, we have investigated whether growth-factor-induced tyrosine phosphorylation is involved in the promotion of cell survival and suppression of apoptosis. Experiments were carried out with the leukemic cell lines HL-60 and M07e and the tyrosine kinase inhibitors genistein and tyrphostin AG82. Both the tyrosine kinase inhibitors induced apoptosis of HL-60 and M07e cells. This was indicated by the appearance of DNA degradation and morphologic evidence of nuclear condensation and fragmentation. It was also confirmed by flow cytometry of DNA, which showed apoptotic cells as a fraction of cells characterized by a diminished DNA stainability, represented on the DNA frequency histograms as a distinct peak below the G0/G1 population. Kinase inhibitors also reduced the fraction of cells in the S phase of the cell cycle. That tyrphostin specifically inhibited tyrosine kinases was further suggested by the prevention of its effects by the tyrosine phosphatase inhibitor sodium orthovanadate (vanadate), at least during the first 18-24 h of treatment. The incomplete prevention of genistein effects by vanadate suggests that genistein is a less specific inhibitor of tyrosine kinases than tyrphostin, and may also act as an inhibitor of topoisomerase II. Vanadate also prevented apoptosis and reduction of the S phase in M07e cells cultured for 24 h in the absence of growth factors. These results suggest that tyrosine phosphorylation is an essential step in IL-3 and GM-CSF signal transduction. Since in our experimental model the effects of tyrosine kinase inhibition and growth factor deprivation could be reversed by concomitant inhibition of tyrosine phosphatases, it is suggested that a balance between tyrosine kinases and tyrosine phosphatases establishes whether a cell will survive or undergo apoptosis.     

Conformation and lipid binding properties of four peptides derived from the membrane-binding domain of CTP:phosphocholine cytidylyltransferase

We are probing the mechanism of the lipid selective membrane interactions of CTP:phosphocholine cytidylyltransferase (CT). We have proposed that the membrane binding domain of CT (domain M) consists of a continuous amphipathic alpha-helix between residues approximately 240-295 [Dunne, S. J., et al. (1996) Biochemistry 35, 11975-11984]. This study examined the secondary structure and membrane binding properties of synthetic peptides derived from domain M: a 62mer peptide encompassing the entire domain (Pep62), a 33mer corresponding to the N-terminal portion (PepNH1), and two 33mers corresponding to the three C-terminal 11mer repeats, one with the wild-type sequence (Pep33Ser), and one with the three serines in the nonpolar face substituted with alanine (Pep33Ala). Peptide secondary structure was analyzed by circular dichroism, and lipid interactions were analyzed by a direct vesicle binding assay, by effects of lipid vesicles on peptide tryptophan fluorescence, and by monolayer surface pressure changes. All peptides bound to vesicles as alpha-helices with selectivity for anionic lipids. Binding involved intercalation of the peptide tryptophan into the hydrophobic membrane core. PepNH1, the peptide with the highest positive charge density, showed strong selectivity for anionic lipids. PepNH1 and Pep33Ser did not bind to PC vesicles; however, the more hydrophobic peptides, Pep33Ala and Pep62, did bind to PC vesicles, with apparent partition coefficients for PC that were only approximately 1 order of magnitude lower than those for PC/PG (1/1). Our results suggest that the polar serines interrupting the nonpolar face of the amphipathic helix serve to lower the lipid affinity and thereby enhance selectivity for anionic lipids. Although diacylglycerol is an activator of the enzyme, none of the peptides responded differentially to PC/diacylglycerol vesicles versus pure PC vesicles, suggesting that domain M alone is not sufficient for the enzyme's response to diacylglycerol. Increases in surface pressure at an air-water interface indicated that the domain M peptides had strong surface-seeking tendencies. This supports a binding orientation for domain M parallel to the membrane surface. Binding of CT peptides to spread lipid monolayers caused surface pressure reductions, suggesting condensation of lipids in the formation of lipid-peptide complexes. At low monolayer surface pressures, Pep62 interacted equally with anionic and zwitterionic phospholipids. This suggests that one determinant of the selectivity for anionic lipids is the lipid packing density (area per molecule).     

Construction of a plasmid carrying both CTP synthetase and a fused gene formed from cholinephosphate cytidylyltransferase and choline kinase genes and its application to industrial CDP-choline production: enzymatic production of CDP-choline from orotic acid (Part II)

A new method for enzymatic production of cytidine diphosphate choline (CDP-choline) from orotic acid and choline chloride was developed. To establish an industrial manufacturing process, we constructed a plasmid, pCKG55, which simultaneously expressed in Escherichia coli the three following enzymes; CTP synthetase (encoded by the pyrG gene from E. coli), cholinephosphate cytidylyltransferase (encoded by the CCT gene from Saccharomyces cerevisiae), and choline kinase (encoded by the CKI gene from S. cerevisiae). CCT and CKI genes on pCKG55 were designed to be expressed as a single CCT/CKI fused protein. This CCT/CKI fused protein retained both activities and the thermal stability of its cholinephosphate cytidylyltransferase activity was nearly the same as the native CCT enzyme. Corynebacterium ammoniagenes KY13505 and E. coli MM294/pCKG55 were cultured in 5-liter jar fermentor independently. Equal volumes of each broth were mixed in a 2-liter jar fermentor, and then the enzymatic reaction was done using 47 mM orotic acid and 60 mM choline chloride as substrates. After 23 h of the reaction at 32 degrees C, 21.5 mM (11 g/liter) of CDP-choline was accumulated.     

Cellular pharmacology of mitoxantrone in p-glycoprotein-positive and -negative human myeloid leukemic cell lines

Previous reports suggest that resistance to mitoxantrone in different tumor cell lines is unrelated to the overexpression of p-glycoprotein. In order to determine the role of p-glycoprotein in the cellular pharmacology of mitoxantrone flow cytometry and confocal microscopy were used to study two human myeloid leukemia cell lines selected for resistance to mitoxantrone (HL-60MX2) and doxorubicin (HL-60DOX). To optimize the detection of intracellular mitoxantrone, we determined the maximum excitation (607 nm) and emission (684 nm) wavelength by fluorescence spectroscopy. The modified flow cytometric conditions using 568.2 nm laser emission for excitation and a 620 nm long pass filter for fluorescence collection resulted in a 1-log increase in sensitivity, compared with standard 488-nm laser excitation. Uptake and retention of mitoxantrone in the presence of verapamil, a calcium channel blocker known to inhibit p-glycoprotein, were analyzed. Our results showed no change in uptake and retention of the drug in p-glycoprotein-negative mitoxantrone-resistant HL-60MX2 cells and in its sensitive parental line, HL-60s. In contrast, 3.1- and 2.4-fold increases were found in uptake and retention of mitoxantrone in p-glycoprotein-positive cells (HL-60DOX) incubated with verapamil. Confocal microscopy of intracellular drug distribution demonstrated reduced nuclear uptake, which could be reversed by verapamil, in HL-60DOX. A characteristic punctate pattern was observed for the intracytoplasmic drug distribution in HL-60DOX and HL-60MX2 cells and was partially modified by the presence of verapamil in HL-60DOX cells. Verapamil increased cytotoxicity of mitoxantrone two-fold in HL-60DOX cells, 1.4-fold in HL-60MX2, and had no effect in HL-60s. Our study demonstrates that the cellular pharmacology of mitoxantrone is affected by p-glycoprotein and can be reversed at least in part by verapamil. Other mechanisms of resistance however, seem to play a determinant role in the modulation of mitoxantrone cytotoxicity.     

Regulation of telomerase activity in immortal cell lines

Telomerase is a ribonucleoprotein whose activity has been detected in germ line cells, immortal cells, and most cancer cells. Except in stem cells, which have a low level of telomerase activity, its activity is absent from normal somatic tissues. Understanding the regulation of telomerase activity is critical for the development of potential tools for the diagnosis and treatment of cancer. Using the telomeric repeat amplification protocol, we found that immortal, telomerase-positive, pseudodiploid human cells (HT1080 and HL60 cells) sorted by flow repressed in quiescent cells. This was true whether quiescence was induced by contact inhibition (NIH 3T3 mouse cells), growth factor removal (bromodeoxyuridine-blocked mouse myoblasts), reexpression of cellular senescence (the reversibly immortalized IDH4 cells), or irreversible cell differentiation (HL60 promyelocytic leukemia cells and C2C12 mouse myoblasts). Taken together, these results indicate that telomerase is active throughout the cell in dividing, immortal cells but that its activity is repressed in cells that exit the cell cycle. This suggests that quiescent stem cells that have the potential to express telomerase may remain unaffected by potential antitelomerase cancer therapies.     

Aromatase activity and the effect of estradiol and testosterone on DNA synthesis in endometrial carcinoma cell lines

Human endometrial and breast carcinoma cell lines were examined for aromatase activity and the effects of sex steroids (estradiol and testosterone) on DNA synthesis. Aromatase activity was high (greater than 500 fmol/10(7) cells/24 h) in the cell lines MCF-7 and OMC-2, moderate (100-499 fmol/10(7) cells/24 h) in the cell lines HEC-59 and Ishikawa, and low (less than 100 fmol/10(7) cells/24 h) in the HHUA cell line. A substantial stimulation of DNA synthesis by estradiol (10(-9)M) was observed in cell lines HEC-59, OMC-2, and MCF-7, with an increase in [3H]thymidine uptake of over 250%. The Ishikawa cell line was stimulated moderately (115-249%). No estradiol-induced increase in DNA synthesis was observed in HHUA. Responsiveness of DNA synthesis to testosterone was observed in cell lines that showed the greatest response to estradiol, namely HEC-59, OMC-2, and MCF-7. Otherwise, estrogen-responsiveness did not always correlated with a significant aromatase activity. These data suggest that some but not all endometrial carcinomas may possess an aromatase-dependent growth stimulating system.