Radio-sensitizing effect of MEK inhibition in glioblastoma in vitro and in vivo.

INTRODUCTION: Glioblastoma (GBM) is an incurable cancer type. New therapeutic options are investigated, including targeting the mitogen-activated protein kinase (MAPK) pathway using MEK inhibitors as radio-sensitizers. In this study, we investigated whether MEK inhibition via PD0325901 leads to radio-sensitization in experimental in vitro and in vivo models of GBM. MATERIALS AND METHODS: In vitro, GBM8 multicellular spheroids were irradiated with 3 fractions of 2 Gy, during 5 consecutive days of incubation with either PD0325901 or MEK-162. In vivo, we combined PD0325901 with radiotherapy in the GBM8 orthotopic mouse model, tumor growth was measured weekly by bioluminescence imaging and overall survival and toxicity were assessed. RESULTS: Regrowth and viability of spheroids monitored until day 18, showed that both MEK inhibitors had an in vitro radio-sensitizing effect. In vivo, PD0325901 concentrations were relatively constant throughout multiple brain areas and temporal PD0325901-related adverse events such as dermatitis were observed in 4 out of 14 mice (29%). Mice that were treated with radiation alone or combined with PD0325901 had significantly better survival compared to vehicle (both P < 0.005), however, no significant interaction between PD0325901 MEK inhibition and irradiation was observed. CONCLUSION: The difference between the radiotherapy-enhancing effect of PD0325901 in vitro and in vivo urges further pharmacodynamic/pharmacokinetic investigation of PD0325901 and possibly other candidate MEK inhibitors.

Technical note: quantification of plasma 1- and 3-methylhistidine in dairy cows by high-performance liquid chromatography-tandem mass spectrometry.

To improve monitoring of protein mobilization in dairy cows, we developed and evaluated a method to quantify 1-methylhistidine and 3-methylhistidine in plasma by HPLC-tandem mass spectrometry. The analytical method described is (1) sensitive: both histidine derivates can be detected in the picomole range; (2) accurate: intra- and interassay coefficients of variation were < 5% for all standard solutions of 1-methylhistidine and 3-methylhistidine measured (31 to 500 pmol); (3) specific: 1-methylhistidine is clearly separated from 3-methyl-histidine in plasma samples from dairy cows; and (4) flexible: can be easily adapted to measure other amino acids or compounds containing a primary amine. 1-Methylhistidine is present in plasma of dairy cows at concentrations of 5.0 ± 1.7 µM, similar to concentrations of 3-methylhistidine (4.4 ± 2.4 µM). Analytical separation of both histidine metabolites is essential when plasma 3-methylhistidine is used as indicator for muscle breakdown in dairy cows. Specific quantification of the concentration of 3-methylhistidine in bovine plasma samples by HPLC tandem mass spectrometry can improve monitoring of protein mobilization in dairy cows.

Protein and fat mobilization and associations with serum ß-hydroxybutyrate concentrations in dairy cows.

The objective of this study was to obtain information on variation between dairy cows in muscle and fat tissue mobilization around parturition and to study the association between protein and fat mobilization and serum ß-hydroxybutyrate (BHBA) concentrations (hyperketonemia) in this period. Thirty-four cows kept under similar conditions at a university dairy farm (no experimental treatments) were monitored from 4 wk before until 8 wk after calving. Mobilization of muscle protein was investigated by analysis of plasma 3-methylhistidine concentrations (3-MH, analyzed by a recently developed HPLC tandem mass spectrometry method) and ultrasound measurements of longissimus muscle thickness. Mobilization of fat tissue was monitored by serum nonesterified fatty acid (NEFA) concentrations and ultrasound measurements of backfat thickness. Large variation was observed between cows in onset and duration of periparturient protein and fat mobilization. Plasma 3-MH concentrations and muscle thickness profiles indicated that protein mobilization started, on average, before parturition and continued until approximately wk 4 of lactation. Serum NEFA concentrations and backfat thickness profiles showed that fat mobilization occurred from parturition until the end of the study. Thus, muscle protein mobilization occurred in advance of fat mobilization in most cows from this study. We hypothesized that this might be due to a prepartum amino acid deficiency in the absence of negative energy balance. The incidence of hyperketonemia in this study was 16/34 = 47%. With the exception of 3 cows defined as having severe hyperketonemia, cows with lower 3-MH concentrations had higher serum BHBA concentrations. A possible explanation for this observation might be that higher mobilization of protein around calving might restrict ketone body production due to the higher availability of glucogenic precursors in the period of most severe negative energy balance and highest fat mobilization. The validity of this hypothesis needs to be confirmed, but data from this study indicate that further research on the role of protein mobilization in the etiology of hyperketonemia in dairy cows is needed.

Iliopsoas impingement after total hip replacement: the results of non-operative management, tenotomy or acetabular revision.

We have reviewed a group of patients with iliopsoas impingement after total hip replacement with radiological evidence of a well-fixed malpositioned or oversized acetabular component. A consecutive series of 29 patients (30 hips) was assessed. All had undergone a trial of conservative management with no improvement in their symptoms. Eight patients (eight hips) preferred continued conservative management (group 1), and 22 hips had either an iliopsoas tenotomy (group 2) or revision of the acetabular component and debridement of the tendon (group 3), based on clinical and radiological findings. Patients were followed clinically for at least two years, and 19 of the 22 patients (86.4%) who had surgery were contacted by phone at a mean of 7.8 years (5 to 9) post-operatively. Conservative management failed in all eight hips. At the final follow-up, operative treatment resulted in relief of pain in 18 of 22 hips (81.8%), with one hip in group 2 and three in group 3 with continuing symptoms. The Harris Hip Score was significantly better in the combined groups 2 and 3 than in group 1. There was a significant rate of complications in group 3. This group initially had better functional scores, but at final follow-up these were no different from those in group 2. Tenotomy of the iliopsoas and revision of the acetabular component are both successful surgical options. Iliopsoas tenotomy provided the same functional results as revision of the acetabular component and avoided the risks of the latter procedure.

Luteinizing hormone inhibits Fas-induced apoptosis in ovarian surface epithelial cell lines.

Gonadotrophins including LH have been suggested to play an important role in the etiology of epithelial ovarian cancers. The goal of the present study was to obtain more insight in the mechanism of gonadotrophin action on ovarian surface epithelium (OSE) cells. As the Fas system is known to be a major player in the regulation of the process of apoptosis in the ovary, we investigated whether LH interfered with Fas-induced apoptosis in the human OSE cancer cell lines HEY and Caov-3. Activation of Fas receptor by an agonistic anti-Fas receptor antibody induced apoptosis, as was evaluated by caspase-3 activation, poly(ADP-ribose) polymerase fragmentation, phosphatidylserine externalization and morphological changes characteristic of apoptosis. Co-treatment with LH reduced the number of apoptotic cells following activation of Fas in a transient manner, while LH by itself did not affect apoptosis or cell proliferation. The anti-apoptotic effect of LH could be mimicked by the membrane-permeable cAMP analog 8-(4-chlorophenylthio) cAMP (8-CPT-cAMP), and blocked by H89, a specific inhibitor of protein kinase A (PKA). In conclusion, these findings suggest that LH protects HEY cells against Fas-induced apoptosis through a signaling cascade involving PKA. Although it is plausible that in vivo LH might also enhance OSE tumor growth through inhibition of apoptosis, further research is necessary to confirm this hypothesis.

Effects of dietary conditions on the pool sizes of precursors of phosphatidylcholine and phosphatidylethanolamine synthesis in rat liver.

We developed a new method for the determination of choline- and ethanolamine-containing precursors of phosphatidylcholine and phosphatidylethanolamine after their separation by HPLC and we have studied the effects of different dietary conditions on the pool sizes of these metabolites in rat liver. Fasting for 48 h induced only a small decrease in the amounts of phosphatidylethanolamine and its water-soluble precursors. Upon refeeding with a high-sucrose, fat-free diet for 72 h, the levels of ethanolamine-containing compounds were only slowly restored. The effects of various dietary conditions on the amounts of phosphatidylcholine and its water-soluble precursors were much more pronounced. Fasting induced a sharp decrease, especially of the amount of cholinephosphate. However, the levels of phosphatidylcholine and the choline-containing precursors were rapidly restored upon refeeding for 24 h. Continued refeeding for an additional 48 h enhanced the cholinephosphate pool size to a level more than double that found in normally fed rats. The latter effect was accompanied by an inhibition of the enzyme CTP:choline-phosphate cytidylyltransferase. The results are discussed in view of a possible regulatory mechanism that may balance the amounts of phosphatidylcholine and phosphatidylethanolamine.

Stimulation of phosphatidylethanolamine synthesis in isolated rat hepatocytes by phorbol 12-myristate 13-acetate.

Incubation of freshly isolated rat hepatocytes in the presence of phorbol 12-myristate 13-acetate stimulates the incorporation of [1,2-14C]ethanolamine into phosphatidylethanolamines. This stimulation is strongly dependent on the ethanolamine concentration in the medium and becomes apparent at ethanolamine concentrations above 25 microM. Treatment of hepatocytes with phorbol 12-myristate 13-acetate results in a decreased labelling of intracellular ethanolamine, ethanolaminephosphate and CDPethanolamine. Exposure of cells to phorbol 12-myristate 13-acetate induces an increase of the activity of the enzymes CTP: ethanolaminephosphate cytidylyltransferase and ethanolaminephosphotransferase. These effects are accompanied by a decrease of the pool size of ethanolaminephosphate and CDPethanolamine and an increase of the level of diacylglycerols after 30 min of incubation in the presence of phorbol 12-myristate 13-acetate. Upon prolonged incubation, the CDPethanolamine and diacylglycerol pools are restored to the level found in untreated cells. These results indicate that stimulation of phosphatidylethanolamine synthesis by phorbol 12-myristate 13-acetate is probably exerted at the level of CTP : ethanolaminephosphate cytidylytransferase, although there may be an additional effect on the subsequent step of phosphatidylethanolamine synthesis, the formation of phosphatidylethanolamines from CDPethanolamine and diacylglycerols.

Induction of hepatocyte proliferation after partial hepatectomy is accompanied by a markedly reduced expression of phosphatidylethanolamine N-methyltransferase-2.

Expression of phosphatidylethanolamine N-methyltransferase (PEMT)-2 in rat hepatoma cells caused an increase in the time for cell division from 18 to 50 h [Cui et al. (1994) J. Biol. Chem. 269, 24531-24533]. We investigated whether or not a similar inverse relationship might exist for liver proliferation in vivo. Thus, partial hepatectomized rats were used to investigate the expression of PEMT2 during liver regeneration. Enhanced biosynthesis of phosphatidylcholine after partial hepatectomy was due to increased activity and amount of CTP:phosphocholine cytidylyltransferase. On the other hand the total activity of PEMT was markedly decreased during the first days of rat liver regeneration. Maximal decrease of total PEMT activity (45%) and loss of PEMT2 protein (90%) coincided with maximal DNA synthesis and CTP:phosphocholine cytidylyltransferase activity 24 h after partial hepatectomy in both male and female rats. Supplementing dietary choline in the diets of female rats shifted this pattern from 24 h to 36 h after partial hepatectomy, whereas the pattern in male rats was not affected. Northern blot studies showed that the amount of PEMT2 mRNA was decreased accordingly, suggesting regulation of the amount and activity of PEMT2 at a pre-translational level. Thus, our data show a reciprocal regulation of CTP:phosphocholine cytidylyltransferase and PEMT2 at the level of gene expression in regenerating rat liver. These results implicate PEMT2 in the regulation of hepatocyte cell growth in a physiologically relevant model.

CTP: phosphocholine cytidylyltransferase is both a nuclear and cytoplasmic protein in primary hepatocytes.

CTP:phosphocholine cytidylyltransferase (CT) has recently been reported to be a predominantly intranuclear enzyme in several cell lines (Wang et al., J. Biol. Chem. 268, 5899-5904 (1993)). This contrasts with previous reports that CT was a cytosolic protein that translocated to the endoplasmic reticulum upon activation. The aim of the present study was to compare the localization of CT in CHO cells and in primary rat hepatocytes. Indirect immunofluorescence of CHO cells revealed a largely nuclear localization of the CT. On the other hand, immunogold electron microscopy and biochemical studies showed a similar density of distribution of CT between the nucleus and cytoplasm. In primary rat hepatocytes immunofluorescence studies indicated that CT was largely cytoplasmic. Studies by immunogold electron microscopy of rat hepatocytes demonstrated that the enzyme was homogeneously distributed throughout all cytoplasmic regions and the nucleoplasm. This result was confirmed by biochemical studies using digitonin and streptolysin O, which permeabilizes the plasma membrane of cells. Enucleation studies indicated that in CHO cells 76% of the CT activity was in the nuclear fraction, whereas in hepatocytes only 32% was recovered in this fraction. The data indicate that CT is found both in nuclear and cytoplasmic fractions of primary hepatocytes and is not predominantly a nuclear enzyme.

Isozymic forms of protein kinase C in regenerating rat liver.

The expression of multiple forms of protein kinase C (PK-C) was studied in regenerating rat liver using hydroxyapatite column chromatography. Two forms of the enzyme were found in the cytosolic as well as membrane fraction of livers from partially hepatectomized rats. The kinetic variation in the activation of these two liver isozymes by fatty acids, phosphatidylserine and diacylglycerol was similar to that reported for the PK-C subspecies from rat brain, designated types II and III. Intracellular redistribution of PK-C caused by phorbol 12-myristate 13-acetate (PMA) was concentration-dependent and was due to translocation of isozyme III, because type II was insensitive to 5 x 10(-8) M PMA. The activity ratio of the two isozymes in either the particulate or cytosolic fraction was the same at 22 h as compared to 4 h after partial hepatectomy.

The effect of 5-thioglucose on the energy metabolism of Schistosoma mansoni in vitro.

5-Thioglucose (5-TG) had a marked effect on the energy metabolism of Schistosoma mansoni in vitro: the conversion of external glucose into lactate by intact worms was severely inhibited. This inhibition of glycolysis was instantaneous, independent of the oxygen concentration and competitive with respect to glucose. Degradation of 0.5 mM external (14C-labelled) glucose was inhibited for 80% in the presence of 20 mM 5-TG. On the other hand the degradation of endogeneous glycogen to lactate was uninhibited. This shows that the inhibition of glucose breakdown occurred at the entrance of glucose into the cell and/or at the hexokinase reaction. It was demonstrated that 5-TG inhibited both the uptake of glucose and the activity of hexokinase. However, it was concluded that in the intact worm 5-TG blocked glycolysis by its competitive inhibition of hexokinase. In intact S. mansoni worms hexokinase is probably the rate-limiting enzyme of glycolysis. Krebs-cycle activity and lactate production do not occur at a fixed ratio: at lower rates of pyruvate formation Krebs-cycle activity was favoured.

Phosphatidylethanolamine methylation and hepatoma cell growth.

Phosphatidylethanolamine is converted to phosphatidylcholine in hepatocytes via the enzyme phosphatidylethanolamine N-methyltransferase (PEMT). An isoform, PEMT2 has been cloned, expressed and localized to a mitochondria-associated membrane in rat liver. Expression of PEMT2 caused a decreased rate of cell division of cultured rat hepatoma cells. Mechanistic studies suggest that the slower growth of transfected hepatoma cells may be due to down regulation of CTP: phosphocholine cytidylyltransferase and the CDP-choline pathway for phosphatidylcholine biosynthesis. A role for PEMT2 in the regulation of hepatocyte cell division is also indicated by PEMT2 down-regulation in regenerating rat liver.

Effects of a single dose of dexamethasone-21-isonicotinate on the metabolism of heifers in early lactation.

Eight Swedish crossbred heifers, about two-and-a-half years old, were given a single intramuscular dose of dexamethasone-21-isonicotinate between nine and 15 days after they had calved and eight similar heifers were left untreated. The treatment had no significant effects on the lipolytic activity of the heifers' fat tissues, and no effect on the concentrations of non-esterified fatty acids and beta-hydroxybutyrate in blood or the triacylglycerol content of the liver. However, there were significant increases in plasma glucose concentrations two days after the injection and in plasma insulin concentrations two and four days after the injection.

8-year survivorship analysis and subjective results of 687 primary Balgrist hip sockets.

The Balgrist hip socket consists of an outer split ring in the form of a truncated cone, made of titanium, which is expanded by a tapered HDPE insert during implantation, thus ensuring firm primary press-fit and the possibility of retightening in the postoperative remodelling phase. Between November 1987 and October 1996, 687 primary Balgrist hip sockets were implanted in 555 patients. Five hundred and thirty-seven patients were investigated. Of these patients, 71.1% never had pain in the operated hip, 88.1% had no problems putting on their shoes, 76.2% were able to walk one or more hours. Furthermore, 91.7% are very or mostly content with the postoperative result. Nineteen hip sockets had to be revised until April 1997. With a 92.1% Kaplan-Meier survivorship rate after 8 years the Balgrist hip socket ranks among the most successful noncemented acetabular components.

Effects of ß-hydroxybutyrate and isoproterenol on lipolysis in isolated adipocytes from periparturient dairy cows and cows with clinical ketosis.

An in vitro model was used to investigate effects of ß-hydroxybutyrate and isoproterenol (ß-adrenergic receptor agonist) on lipolysis in isolated adipocytes from late pregnant and recently calved dairy cows (n=5) and cows with clinical ketosis (n=3). Incubation with 3.0 mmol/L ß-hydroxybutyrate reduced lipolysis in isolated adipocytes. This inhibitory effect was lower in the first lactation week (47%±16%) compared with late pregnancy (71%±6.5%). Incubation with 0.3 µmol/L isoproterenol stimulated lipolysis in isolated adipocytes from periparturient dairy cows. Basal lipolysis resulted in non-esterified fatty acid to glycerol ratios in the incubation media of 2.0±0.23 in prepartum samples, 2.1±0.23 in the first lactation week and 2.2±0.09 in cows with clinical ketosis. ß-Hydroxybutyrate reduced lipolysis by 45%±9.6% in isolated adipocytes from cows with clinical ketosis, indicating that impaired feedback of ß-hydroxybutyrate may not play a role in the disease etiology.

Stimulation of CTP:phosphocholine cytidylyltransferase by free cholesterol loading of macrophages involves signaling through protein dephosphorylation.

Free cholesterol-loaded macrophages in atheromata synthesize excess phosphatidylcholine (PC), which may be an important adaptive response to the excess free cholesterol (FC) load. We have recently shown that FC loading of macrophages leads to 2-4-fold increases in PC mass and biosynthesis and to the post-translational activation of the membrane-bound form of CTP:phosphocholine cytidylyltransferase (CT), a key enzyme in PC biosynthesis. Herein, we explore further the mechanism of CT activation in FC-loaded macrophages. First, enrichment of membranes from control macrophages with FC in vitro did not increase CT activity, and PC biosynthesis in vivo is up-regulated by FC loading even when CT and FC appear to be mostly in different intracellular sites. These data imply that FC activates membrane-bound CT by a signaling mechanism. That the proposed signaling mechanism involves structural changes in the CT protein was suggested by data showing that two different antibodies against synthetic CT peptides showed increased recognition of membrane-bound CT from FC-loaded cells despite no increase in CT protein. Since CT is phosphorylated, two-dimensional maps of peptides from 32P-labeled control and FC-loaded macrophages were compared: six peptide spots from membrane-bound CT, but none from soluble CT, were dephosphorylated in the FC-loaded cells. Furthermore, incubation of FC-loaded macrophages with the phosphatase inhibitor, calyculin A, blocked increases in both PC biosynthesis and antipeptide-antibody recognition of CT. Last, treatment of membranes from control macrophages with lambda phage protein phosphatase in vitro increased both CT activity (2-fold) and antipeptide-antibody recognition of CT; soluble CT activity and antibody recognition were not substantially affected by phosphatase treatment. In summary, FC loading of macrophages leads to the partial dephosphorylation of membrane-bound CT, and possibly other cellular proteins, which appears to be important in CT activation. This novel regulatory action of FC may allow macrophages to adapt to FC loading in atheromata.

Phosphorylation and activation of the arachidonate-mobilizing phospholipase A2 in macrophages in response to bacteria.

The role of potential target enzymes in the protein-kinase-C-independent eicosanoid response triggered by certain bacteria in murine peritoneal macrophages [Svensson, U., Holst, E. & Sundler, R. (1991) Eur. J. Biochem. 202, 699-705] has been investigated. The eicosanoid response was found to be due to an increase in the mobilization of arachidonate rather than to inhibition of arachidonate esterification or activation of the cyclooxygenase pathway and to be accompanied by a persistent increase in the activity of the arachidonate-mobilizing phospholipase A2 (PLA2-85). Also, down-regulation of protein-kinase C by prolonged treatment with 4 beta-phorbol 12-myristate 13-acetate did not reduce the bacterial activation of PLA2-85. The increase in activity of PLA2-85, like the increase in eicosanoid formation, showed a lag period of approximately 10 min. Furthermore, exposure of 32P-labeled macrophages to either bacteria (Gardnerella vaginalis) or the protein-phosphatase inhibitor okadaic acid caused an increase in the phosphorylation of PLA2-85. Okadaic acid (0.5 microM), which itself caused arachidonate mobilization and activation of PLA2-85 after a lag period of approximately 45 min, greatly promoted the response to bacteria even at earlier time points. This study provides strong evidence that the eicosanoid response to bacteria in macrophages occurs via a protein-kinase-C-independent activation of PLA2-85 and that this activation is due to an increase in the phosphorylation of the enzyme.

Expression of phosphatidylethanolamine N-methyltransferase-2 cannot compensate for an impaired CDP-choline pathway in mutant Chinese hamster ovary cells.

Phosphatidylcholine is a product of the CDP-choline pathway and the pathway that methylates phosphatidylethanolamine. We have asked the question: are the two pathways functionally interchangeable? We addressed his question by investigating the expression of phosphatidylethanolamine N-methyltransferase-2 (PEMT2) of rat liver in mutant Chinese hamster ovary cells (MT-58) (Esko, J. D., Wermuth, M.M., and Raetz, C. R. H. (1981) J. Biol. Chem. 256, 7388-7393) defective in the CDP-choline pathway for phosphatidylcholine biosynthesis. Cell lines stably expressing different amounts of PEMT2 activity (up to 700 pmol/min.mg protein) were isolated. A positive correlation between the amount of PEMT2 activity expressed and the incorporation of [3H]methionine into phosphatidylcholine at both the permissive and restrictive temperatures showed that PEMT2 was functional in the Chinese hamster ovary MT-58 cells. In contrast to mutant cell lines stably expressing transfected CTP:phosphocholine cytidylyltransferase, the cell lines stably expressing PEMT2 did not survive at the restrictive temperature. Determination of the phosphatidylcholine mass in wild type cells, mutant MT-58 cells, and cells with the highest level of PEMT2 expression showed that PEMT2 was functional and synthesized the same amount of phosphatidylcholine as did wild type cells at the restrictive temperature. Indirect immunofluorescence studies showed that localization of the over-expressed cytidylyltransferase in MT-58 cells was largely nuclear, whereas PEMT2 was predominantly located outside the nucleus. Our data show that methylation of phosphatidylethanolamine to phosphatidylcholine cannot substitute for the CDP-choline pathway.

Metabolic responsiveness to phorbol ester and activity of protein kinase C in isolated hepatocytes from partially hepatectomized rats.

The ability of isolated rat hepatocytes to respond to phorbol-12-myristate-13-acetate (PMA) with acute stimulation of de novo fatty acid synthesis was markedly depressed at 4, 22 and 48 h after partial hepatectomy (PH). This desensitization was not due to surgical stress as shown by comparison with hepatocytes from sham-operated animals. Moreover, the total activity of protein kinase C (PK-C), the principal phorbol ester receptor, was not down-regulated at 22 h after partial hepatectomy. Partial hepatectomy rather caused a small but distinct shift in subcellular PK-C distribution toward the particulate fraction thereby suggesting a modest activation of PK-C. We conclude that the PH-induced desensitization to PMA occurs at a point beyond PK-C activation.

Expression of phosphatidylethanolamine N-methyltransferase-2 in McArdle-RH7777 hepatoma cells inhibits the CDP-choline pathway for phosphatidylcholine biosynthesis via decreased gene expression of CTP:phosphocholine cytidylyltransferase.

Phosphatidylethanolamine N-methyltransferase-2 (PEMT2) of rat liver was expressed in McArdle-RH7777 rat hepatoma cells, which lack endogenous PEMT activity. Expression of the enzyme was confirmed by assay of PEMT activity and immunoblotting. There was no change in the amount of phosphatidylcholine in the transfected cells [Cu, Houweling and Vance (1994) J. Biol. Chem. 269, 24531-24533], even though the expression of PEMT2 caused an increased incorporation of [methyl-3H]methionine and [3H]ethanolamine into phosphatidylcholine. In contrast, [3H]serine incorporation into phosphatidylcholine was only marginally enhanced by PEMT2 expression. Incorporation of [methyl-3H]choline into phosphatidylcholine was decreased by greater than 60%, suggesting that the CDP-choline pathway was inhibited as a result of PEMT2 expression. CTP:phosphocholine cytidylyltransferase (CT) activities in transfected cell lines were decreased in proportion to the level of expression of PEMT2. Immunoblot analyses showed a decrease in CT mass as a function of PEMT2 expression. In contrast, there was no change in the mass of protein disulphide-isomerase or the relative amounts of most proteins expressed in the PEMT2-transfected, compared with control, cells. Similarly, the expression of CT mRNA was decreased in PEMT2-expressing cells, whereas the mRNAs for protein disulphide-isomerase and actin were unchanged. When cell growth was slowed by incubating McArdle-RH7777 cells at 25 degrees C, compared with 37 degrees C, there was no difference in the specific activity of the CT. These results argue that PEMT2 expression down-regulates the CDP-choline pathway by decreasing the expression of the gene for the CT. The decreased activity of the CDP-choline pathway might contribute to the slower rate of cell division in PEMT2-transfected hepatoma cells.