Long non-codingRNA (lncRNA) TUG1 and the prognosis of cancer: a meta-analysis.

Some studies assessed the association between lncRNA taurine-upregulated gene 1 (TUG1) and the survival in cancer. However, the results were inconclusive.  Therefore, we performed a meta-analysis to determine this association. We used the following electronic databases to search for eligible literature: PubMed, Embase, Chinese National Knowledge Infrastructure (CNKI) and Wanfang. We used ORs and 95% CIs to measure the association between TUG1 and the survival of cancer. There was no significant association between TUG1 and OS of cancer (HR=1.26, 95% CI=0.97-1.64). In the subgroup analysis by cancer type, significant association could be find in osteosarcoma (HR=1.72, 95% CI=1.27-2.32) and digestive system's tumors (HR=1.66, 95% CI=1.04-2.66). In conclusion, this meta-analysis study indicated that TUG1 might associate with the OS of osteosarcoma and digestive system's tumors.

Expression of mTOR and its inhibitory effect on cell proliferation and apoptosis of breast cancer cells.

The aim of this study is to investigate the expression of mTOR in breast cancer and observe the effect of CCI-779 on proliferation and apoptosis of MDA-MB-231 cells. Immunohistochemical staining was used to detect the expression of mTOR protein in breast cancer tissues and MDA-MB-231 cells. MTT assay was used to assess the effect of CCI-779 on proliferation of MDA-MB-231 cells. Annex-inV-FITC/ PI assay was utilized to evaluate the effect of CCI-779 on apoptosis of MDA-MB-231 cells. Among the 71 cases of breast cancer tissues, 54.9% were mTOR-positive that exhibited significantly higher expression than the 32 cases of normal tissues (21.9%); mTOR protein was also found to be expressed in MDA-MB-231 cells. The mTOR inhibitor CCI-779 significantly inhibited the proliferation of MDA-MB-231 cells that was dose- and time-dependent. However, CCI-779 was unable to induce apoptosis of MDA-MB-231 cells as demonstrated with AnnexinV-FITC/PI assay. mTOR plays a key role in the initiation and development of breast cancer, and its inhibitor CCI-779 exerts a strong suppressive activity against MDA-MB-231 cells, suggesting its therapeutic potential to treat breast cancer.

Developmental differences in activation of cholinephosphate cytidylyltransferase by lipids in rabbit lung cytosol.

Lung cytosolic cholinephosphate cytidylyltransferase is activated by lipids. We examined the lipid activation pattern as a function of development in rabbit lung from 27 days gestation through term (31 days) and in the adult. The enzyme in both the fetal and adult cytosol was dependent on lipids for activity. Extraction of the cytosol with acetone/butanol virtually abolished cytidylyltransferase activity, but the activity could be restored on addition of lipids extracted with chloroform/methanol from additional cytosol. Cytosolic phospholipids from the fetal lung reactivated cytidylyltransferase but both neutral lipids and phospholipids from the adult were required. The lipids had the same effect on cytidylyltransferase activity in delipidated cytosol from either the fetus or adult so the difference in activation pattern was attributable to the lipids rather than the protein. There was a shift from the fetal to the adult lipid activation pattern as development progressed. Further, there was a significant correlation between cytidylyltransferase activities in intact cytosols from developing lung and activities in delipidated cytosol in the presence of lipids from the same animals. Although these data suggest that lipids regulate cytosolic cytidylyltransferase activity in developing lung their physiological significance remains to be established.

Stimulation of cholinephosphate cytidylyltransferase activity by estrogen in fetal rabbit lung is mediated by phospholipids.

We have investigated the mechanism by which estrogen stimulates phosphatidylcholine synthesis in fetal rabbit lung. The hormone increased the activity of cholinephosphate cytidylyltransferase in the 105 000 X g supernatant fraction but had no effect on the activities of this enzyme in the homogenate or other subcellular fractions. Although microsomal cytidylyltransferase has been reported to regulate phosphatidylcholine synthesis in other systems, and translocation of the enzyme from cytosol to microsomes has been reported in association with increased phosphatidylcholine synthesis, we found no evidence of this in the case of estrogen-stimulated phosphatidylcholine synthesis in the fetal lung. Cytosolic cytidylyltransferase activity was dependent on phospholipids. Extraction with acetone/butanol drastically reduced its activity as well as the stimulatory effect of estrogen. The activity and the effect of estrogen were restored on re-addition of lipids extracted with chloroform/methanol from additional supernatants. Fractionation of the total lipids revealed that the stimulatory effect was entirely associated with the phospholipids; neutral lipids and glycolipids did not stimulate. Treatment of the phospholipid fraction with phospholipase C abolished the stimulatory effect. The stimulatory effect of estrogen, however, could not be attributed to any individual phospholipid species but appeared to require the entire phospholipid mixture. We conclude that estrogen stimulates fetal lung phosphatidylcholine synthesis by increasing the activity of cytosolic cytidylyltransferase and this activation in turn is mediated by cytosolic phospholipids.

Stimulation of phosphatidylcholine synthesis by exogenous phosphatidylglycerol in primary cultures of type II pneumocytes.

We examined the effect of exogenous phospholipids on phosphatidylcholine synthesis in primary cultures of adult rat type II pneumocytes. Incubation of the cells with 10 microM phosphatidylglycerol for 2 h stimulated the rate of [3H]choline and [3H]glycerol incorporation into phosphatidylcholine by 72% and 50%, respectively. The effect appeared to be specific for phosphatidylcholine synthesis and was largely on the unsaturated species. Synthesis of disaturated phosphatidylcholine was little stimulated. The stimulatory effect of the lipid is unlikely to be a consequence of increased substrate, since it was not mimicked by glycerol, glycerol 3-phosphate or palmitic acid. Neither does it appear to be due to increased cell growth, since rates of protein and DNA synthesis were not increased. The relevance of these findings to surfactant turnover and reutilization warrants investigation.

I. Suppression by compound 48/80 of bacterial endotoxin-inducible monocytic tissue factor activity: direct blockade of factor VII binding to THP-1 monocytes.

Hypercoagulation with upregulated monocytic tissue factor (TF) activity often occurs under a variety of inflammatory conditions including endotoxemia. The antagonism to bacterial endotoxin (LPS) signaling often results in the depression in TF upregulation. We herein report that compound 48/80 (48/80) significantly depressed LPS-induced TF activity in human and cebus monkey peripheral blood monocytes. Employing a model monocyte-like cell line (THP-1), we explored the regulatory mechanism to identify the inhibitory site(s) of 48/80. We determine whether the inhibition results from the blockade of LPS signaling. 48/80 dose-dependently inhibited LPS-induced TF activity. Chase of LPS-challenged cells with 48/80 also significantly offset TF upregulation. In immunofluorescent approaches, FACScan analysis revealed that 48/80 had no effect on either LPS recognition or the expression of its receptors (CD14 and CD11b). Moreover, LPS-induced TF expression as well as synthesis remained unaffected in the presence of 48/80. Consistent with the independence of LPS action, 48/80 was also able to inhibit TF activity induced by A23187, ionomycin, or Quin-2 AM. Interestingly, 48/80 significantly decreased the FVII binding to either resting or LPS-challenged cells. In conclusion, our results elucidate that the inhibitory action of 48/80 was independent of LPS signaling including recognition, receptor expression, and the induced TF expression/ synthesis. However, 48/80 was able to directly block FVII binding to monocytic TF, thereby resulting in such antagonism to LPS-induced TF-initiated extrinsic coagulation.

Glucocorticoid stimulation of choline-phosphate cytidylyltransferase activity in fetal rat lung: receptor-response relationships.

A number of previous studies using in vivo and cultured fetal lung models have shown that the activity of choline-phosphate cytidylyltransferase, the enzyme which catalyzes a rate-limiting reaction in de novo phosphatidylcholine synthesis, is increased by glucocorticoids and other hormones which accelerate fetal lung maturation. To examine the mechanism of this glucocorticoid action further, we examined the effect of dexamethasone on cytidylyltransferase activity in cultured fetal rat lung explants and related it to specific dexamethasone binding. Dexamethasone stimulated cytidylyltransferase activity in the homogenate, microsomal and 105,000 X g supernatant fractions. The hormone did not alter the subcellular distribution of the enzyme, however; the bulk of the activity was in the supernatant fraction in both the control and dexamethasone-treated cultures. The dose-response curves for stimulation of cytidylyltransferase activity in the supernatant fraction and specific nuclear binding of dexamethasone were similar and both plateaued at approx. 20 nM. The EC50 for cytidylyltransferase stimulation was 6.6 nM and the Kd for dexamethasone binding was 6.8 nM. The relative potencies of various steroids for stimulating choline-phosphate cytidylyltransferase and for specific nuclear glucocorticoid binding were the same: dexamethasone greater than cortisol = corticosterone = dihydrocorticosterone greater than progesterone. The stimulation by dexamethasone of cytidylyltransferase activity and of choline incorporation into phosphatidylcholine were both abolished by actinomycin D. These data show that the stimulatory effect of dexamethasone on fetal rat lung choline-phosphate cytidylyltransferase activity is largely on the enzyme in the supernatant fraction and does not involve enzyme translocation to the microsomes as has been reported for cytidylyltransferase activation in some other systems. This effect of dexamethasone is a receptor-mediated process dependent on RNA and protein synthesis.

Blockade by ruthenium red of tissue factor-initiated coagulation.

The ability of ruthenium red (RuR) to inhibit tissue factor (TF)-initiated blood coagulation was demonstrated at the protein and cellular levels as well as in human plasma. In a single-stage clotting assay, RuR concentration-dependently inhibited rabbit brain thromboplastin (rbTF)-induced coagulation and offset bacterial endotoxin (LPS)-induced monocytic TF (mTF) hypercoagulation; the IC(50)s were estimated at 7.5 and 12.3 microM, respectively. A 15-min preincubation of RuR with rbTF or monocyte suspension resulted in the pronounced inhibition with a significantly lowered IC(50) at 1.8 or 7.7 microM for rbTF or mTF procoagulation, respectively. The differences in IC(50)s between rbTF and mTF without or with the preincubation indicated that TF was a primary target for RuR action. The effect of RuR on the physiological function of TF in FVII activation was demonstrated by the proteolytic cleavage of FVII zymogen to its active forms of serine protease on Western blotting analyses. RuR readily blocked TF-catalyzed FVII activation (diminished FVIIa formation), thus down regulating the initiation of blood coagulation. Inclusion of RuR into human plasma samples in vitro significantly prolonged prothrombin time, indicating the depressed coagulation. FVII activity was inhibited by 30 - 60% depending on the dose; as a result, FX activity also decreased. However, RuR showed no effect on thrombin time. Thus, RuR inhibited FVII activation to block the initiation of coagulation.

Up-regulation by human recombinant transforming growth factor beta-1 of collagen production in cultured dermal fibroblasts is mediated by the inhibition of nitric oxide signaling.

BACKGROUND: Hypertrophic scarring remains the most disabling sequela for burn survivors. Little is known about its pathogenesis. Collagen accumulation, however, has been consistently observed in burn hypertrophic scars (HS). STUDY DESIGN: We have studied collagen production in the dermal fibroblasts derived from HS, which has developed for 9 months to 2 years. Reconstructive surgery was performed to remove HS from which the fibroblasts were cultured. Similarly, the normal cells were grown from the patient's donor site (DS), which provided autografting to the HS site. Collagen production in HS and DS fibroblasts was compared and analyzed in minimal essential amino acid medium containing 5% fetal bovine serum with inclusion of L-ascorbic acid (100 microg/mL) and beta-aminopropoinitrile (100 microg/mL) by monitoring a 20-h [3H]proline incorporation into bacterial collagenase III-digestible protein in the conditioned media. RESULTS: We failed to detect any significant difference in collagen production in vitro between HS and DS. Irrespective of the fibroblasts from HS or DS, collagen production was substantially stimulated by human recombinant transforming growth factor beta-1 (TGF-beta1) (20 ng/mL) by approximately 250% after a 3-day pretreatment. In contrast, sodium nitroprusside (SNP) at 100 microM exhibited significant suppression (68%), which was rescued by hemoglobin (10 microM). TGF-beta1 significantly decreased nitric oxide (NO) production by 55%. In contrast, NO level drastically increased by 350% following SNP treatment. Epidermal growth factor showed no effect on either collagen production or NO level. The linear regression analysis shows a significant inverse correlation (r = 0.72; p < 0.05) of NO level with collagen production, suggesting the involvement of NO signaling in the modulation of collagen production. Consistent with the notion, we further showed that N-nitro-L-arginine methyl ester (100 microM) caused a synergistic stimulation and an arrested inhibition of collagen production in the presence of TGFbeta-1 and SNP, respectively. 8-BrcGMP (300 microM) mimicked the NO inhibitory action, while methylene blue (50 microM) restored the collagen production which was inhibited by SNP. Moreover, 8-BrcGMP offset the stimulation of collagen production. CONCLUSIONS: The dermal fibroblasts derived from HS were not different from normals with respect to collagen production and their responses to regulations. The inhibition of collagen production was achieved by a cGMP-dependent NO action. TGFbeta-1 inhibited NO/cGMP signaling to ensure its stimulatory effect on collagen production in the dermal fibroblasts.

Estrogen stimulation of surfactant synthesis.

Administration of 17 beta-estradiol to pregnant rabbits accelerates fetal lung maturation and stimulates surfactant production: the hormone increases the amount of surfactant in fetal lung lavage, increases the rate of phosphatidylcholine synthesis, depletes fetal lung glycogen, and accelerates morphologic maturation of the fetal lung. Both estrogens and glucocorticoids stimulate fetal lung cholinephosphate cytidylyltransferase in a number of in vivo and in vitro systems and there is increasing evidence that this enzyme may be of particular importance in the regulation of phosphatidylcholine synthesis. Estrogen appears to increase the catalytic activity rather than the amount of cholinephosphate cytidylyltransferase. This action of estrogen is mediated by phospholipids.

Inhibitors of sterol synthesis. A major role of chylomicrons in the metabolism of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one in the rat.

The metabolism of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one (I), a potent regulator of cholesterol (Chol) metabolism which has significant hypocholesterolemic activity upon oral administration to animals, has been investigated in male rats. After intragastric administration of [2,4-3H] I and [4-14C]Chol in triolein to intestinal lymph duct-cannulated rats, most of the 3H of the lymph was associated with chylomicrons. Most of the 3H in the chylomicrons was associated with fatty acid esters of I and the oleate ester represented the major species of the esters of I. After intravenous injection of the isolated doubly-labeled chylomicrons to intact rats, rapid clearance of 3H and 14C from blood was observed which was associated with a rapid and selective uptake of 3H and 14C by liver. The rate of disappearance of 3H from blood and the rate of uptake of 3H by liver were similar, if not identical, to those for 14C. In contrast, the disappearance of 3H from the liver was much more rapid than that of 14C. Studies of the distribution of 3H in liver demonstrated rapid formation of free I and the formation of [3H]Chol. In addition, significant amounts of the 3H in liver were associated with polar materials, a finding which was not observed in the case of 14C. After intravenous administration of the doubly-labeled chylomicrons to bile duct-cannulated rats, very rapid and substantial metabolism of the administered 3H to polar biliary metabolites was observed. The bulk of the 3H not recovered in bile at 49 h after the injection of the labeled chylomicrons was recovered in blood and tissues and almost all (integral of 94%) of this material was associated with Chol and Chol esters. The combined results indicate an important role for chylomicrons in the overall metabolism of I. The selective delivery of I to liver as its oleate ester in chylomicrons (or, more probably, as chylomicron remnants) and the subsequent metabolism of the oleate ester of I in liver has important consequences with respect to the actions of I which are discussed herein.

Bacterial lipopolysaccharide induces phosphatidylcholine breakdown in human leukaemia monocytic U937 cells.

We investigated the effect of bacterial lipopolysaccharide (LPS) on phospholipid (PL) turnover in human monocytic leukaemia U937 cells. Cells were pre-labelled with [3H]choline, [14C]ethanolamine and [3H]inositol for 24 h. By monitoring the radiolabel association with cellular PL, the data indicated that LPS (10 micrograms/ml) drastically altered the catabolism of choline-containing PL; it induced their breakdown by 50% within 20 min. The reutilization of choline or its phosphates for PL synthesis was also suggested as a result of regaining radiolabel in the next 40 min. Choline-containing PL then underwent a second degradation after 60 min; 50% decline in radiolabel was detected at 120 min. In contrast, LPS did not induce the breakdown of phosphatidylethanolamine and phosphatidylinositol through phospholipase C/phospholipase D (PLC/PLD). No significant redistribution of the radiolabel in PL was detected in any cases during chasing. The data clearly indicate that LPS stimulates phosphatidylcholine breakdown, implying that the liberation of phosphatidic acid or diacylglycerol via PLC/PLD reaction may be relevant to the initiation of LPS-induced monocytic activation.

Novel anticoagulant activity of polybrene: inhibition of monocytic tissue factor hypercoagulation following bacterial endotoxin induction.

The enhanced extrinsic coagulation in response to inflammation could contribute to disseminated intravascular coagulation, often manifesting cardiovascular complications. The complex mechanism remains unclear. Nor is the effective anticoagulation well established. The search for arresting hypercoagulation is of antithrombotic relevance. The ability of polybrene (PB) to inhibit tissue factor (TF)-initiated extrinsic blood coagulation was demonstrated at the protein and cellular levels as well as in human plasma samples. In a single-stage clotting assay, PB dose-dependently offset bacterial endotoxin (lipopolysaccharide)-induced monocytic TF (mTF) hypercoagulation and inhibited rabbit brain thromboplastin (rbTF) procoagulation. Consistent with these findings, the significantly prolonged prothrombin time indicated the depressed extrinsic coagulation by PB. However, PB showed no effect on thrombin time. We dissected the extrinsic pathway to further determine the inhibitory site(s) of PB. A two-stage chromogenic assay monitoring S-2288 hydrolysis showed that PB readily blocked mTF-dependent or rbTF-dependent FVII activation, which was verified by the diminished activated factor VII (FVIIa) formation derived from the proteolytic cleavage of its zymogen factor VII on Western blotting analyses. PB had no effect on FVIIa and activated factor X amidolytic activity. Nor was the dissected TF/FVIIa-catalyzed factor X activation affected. In conclusion, the preferential downregulation of factor VII activation was responsible for the depressed extrinsic coagulation. PB could present a novel anticoagulant antagonizing the extrinsic hypercoagulation for the prevention of thrombotic complication following sepsis and inflammations.

Fatty acid unsaturation increases expression and capping of murine lymphocyte CD44 and CD45.

We studied the effect of incubating murine lymphocytes with cis-unsaturated fatty acids on expression and capping of CD44 and CD45. Lymphocytes were incubated with stearic (18:0) or oleic (18:1 omega-9) acid bound to bovine serum albumin (BSA). After incubation with rat anti-CD44 or anti-CD45 monoclonal antibodies and then with fluorescent-labeled anti-rat antibody, mean fluorescence intensity (FI) was measured by using flow cytometry. Capping was measured after warning and fixation in paraformaldehyde. Steady-state fluorescence anisotropy (rs) was measured after the cells had been incubated with trimethylammoniumdiphenylhexatriene. Incubation with oleic acid, but not stearic acid or BSA alone, was associated with an increase in FI of CD44. Expression of CD45, however, was increased by both stearic and oleic acids to the same degree over BSA controls. CD44 and CD45 capping were both increased by incubation with oleic acid. Rs was decreased in cells incubated with oleic acid, suggesting an increase in membrane fluidity. We conclude that incubation with oleic acid increases expression of CD44 and increases capping of both CD44 and CD45. These findings were confirmed in feeding experiments, in which rs was reduced and CD44 capping increased by polyunsaturated fatty acid diets.

Dual-source, dual-energy multidetector CT for the evaluation of pancreatic tumours.

OBJECTIVE: To investigate the potential diagnostic value of dual-energy CT (DECT) with virtual non-enhanced (VNE) and iodine-only images, and to determine the optimal mixed ratio of blended images for evaluation of pancreatic diseases. METHODS: Multiphasic DECT was performed in 44 patients with focal pancreatic disease. DECT was used during the pancreatic and hepatic venous phases, and a peak kilovoltage of 120 kVp was used for both non-contrast phases. For qualitative analysis of the CT images, two radiologists assessed three image sets (VNE, iodine-only and blended images) in order to determine the acceptability of VNE in replacing true non-enhanced (TNE) images, the added value of iodine-only images and the preferred blending ratio. For quantitative analyses, the CT numbers and image noise of the pancreatic parenchyma, lesions, aorta and psoas muscle were measured. The contrast-to-noise ratio of the lesion was calculated on the pancreatic phase images. The effective radiation dose for DECT and TNE images was calculated. Statistical comparisons were made using the Friedman test, the Wilcoxon test, the paired t-test and repeated measures of analysis of variation with Bonferroni correction for multiple comparisons. RESULTS: The level of acceptance of the VNE images in replacing TNE images was 90.9%. Regarding the iodine-only images, 50% of the cases were found to have an added value. The linear-blended images with a weighting factor of 0.5 were preferred. CONCLUSIONS: DECT was able to provide high-quality VNE images that could replace TNE images and iodine-only images showing an added value. Blended images with a weighting factor of 0.5 were preferred by the reviewers.

Inhibition of endotoxin-induced monocytic procoagulant activity by n-alcohols and anesthetics.

1. The effect of n-alcohols (methanol and ethanol) and anesthetics (lidocaine, thiopental, methohexital and thiamylal) on procoagulant activity (PCA) in human peripheral-blood monocytes and non-adherent cultured leukemia promonocytic U937 and THP-1 cells was examined herein. 2. Exposure of whole blood to ethanol showed no effect on PCA in human monocytes. However, ethanol dose-dependently inhibited LPS-induced PCA in isolated human monocytes. 3. In THP-1 cells, ethanol had no significant effect on PCA in either non-challenged or LPS-induced status. However, the induction of PCA by LPS was substantially inhibited when cells were pretreated with 1% ethanol (v/v) for 72 hr. 4. In U937 cells, n-alcohols and anesthetics resulted in dose-dependent depressions in PCA. Importantly, the percent reduction in LPS-induced PCA was much more pronounced than that in non-challenged PCA. 5. These data clearly suggest that n-alcohols and anesthetics readily inhibit the LPS-stimulatory action on monocytic PCA.

Mechanism by which ethanol inhibits phosphatidylcholine biosynthesis in human leukemic monocyte-like U937 cells.

A previous study showing that ethanol (ETOH) blocked [3H]choline incorporation into phosphatidylcholine (PC) suggested an inhibition of PC biosynthesis in human leukemic monocyte-like U937 cells. The mechanism of the inhibitory action of ETOH was investigated. Cells were pulsed with [3H]choline for 30 min and chased in the presence or absence of ETOH for up to 6 h. PC biosynthesis was inhibited drastically within 1 h after exposure to ETOH which increased intracellular cAMP appreciably. After a 3-h treatment, ETOH significantly inhibited both choline kinase (CK) and the cytosolic CTP: cholinephosphate cytidylyltransferase (CT). The inactivated CT was no longer stimulated by exogenous phosphatidylglycerol (PG). There was no evidence for redistribution of CT activity between cytosol and microsomes. When cells were exposed to 8-Bromo-cAMP ranging from 100 to 300 microM, PC biosynthesis remained unaffected despite the drastically elevated cAMP. These results seem to suggest that the raised cAMP is not a prerequisite for the inhibition of PC biosynthesis in U937 cells. Following pretreatment with protein kinase inhibitors (H-89 and K-252a), PC biosynthesis was decreased significantly and the inhibitory effect of ETOH was potentiated. Taken together, our results suggest that the inhibition of PC biosynthesis and the inhibitory effect of ETOH are independent of the activation of cAMP-dependent protein kinase. Unlike protein kinase inhibitors, pretreatment with tyrosine kinase inhibitors (erbstatin, genistein and tyrphostin 25) resulted in differential effects on PC biosynthesis and on the inhibitory action of ETOH. Genistein stimulated PC biosynthesis by 30 per cent as well as partially preventing/reversing the ETOH action, while tyrphostin 25 produced a synergistic inhibition. The relevance of tyrosine phosphorylation/dephosphorylation to the regulation of PC biosynthesis and ETOH action remains to be established.

Differential regulations of phosphatidylcholine biosynthesis in U937 cells by inhibitors of protein and tyrosine kinases.

1. The differential effects of inhibitors of protein kinase (PK) or tyrosine kinase (TK) on phosphatidylcholine (PC) biosynthesis in monocyte-like U937 cells were compared in pulse-chase-studies in which the cells prelabelled with [3H]choline for 30 min were chased in the absence or presence of kinase inhibitors. 2. PKA inhibitor (H-89) decreased the label incorporation into PC, while PKA activator (8-BrcAMP) had no effect. 3. PKC inhibitors (chelerythrine and hypericin) inhibited PC biosynthesis; on the other hand, PKC activator (SC-10) was stimulatory. 4. The inhibition of PC biosynthesis by H-89 and chelerythrine was accompanied by the inactivation of CTP: cholinephosphate cytidylyltransferase (CT). 5. In contrast, TK inhibitor (genistein) markedly stimulated CT and PC biosynthesis, while erbstatin and tyrphostin No. 25 showed no effect.

Stimulation of phosphatidylcholine biosynthesis by hemicholinium-3, a potent inhibitor of choline uptake in human leukemic monocyte-like U937 cells.

The effect of hemicholinium-3 (HC-3) on choline uptake and phosphatidylcholine (PC) biosynthesis was examined in human leukemic monocyte-like U937 cells. HC-3 inhibited [3H]choline uptake in a dose- and time-dependent manner. After a 3 h treatment, HC-3 (100 microM) decreased choline uptake by as much as 80 per cent (p < 0.0001; n = 4). Reduction of incorporation of label into PC was also detected in a dose-dependent manner; the extent of inhibition, however, was always 10-20 per cent less than that observed in the total uptake. At 3 h HC-3 decreased the incorporation into PC by 65 per cent (p < 0.0001; n = 5). Kinetic studies in vivo showed that HC-3 inhibited total uptake and incorporation into PC differently, suggesting that the labelling of PC is not simply dictated by [3H]choline uptake. In separate experiments, cells were pretreated with 100 microM HC-3 for 3 h. After washing, the inhibitory effect on total uptake was no longer observed, while a 20 per cent stimulation of the incorporation into PC was obtained in these pretreated cells. In pulse-chase studies, the cells were prelabelled with [3H]choline for 30 min and chased with HC-3 for up to 3 h; the results showed a significant stimulation of incorporation into PC in a longer chase with 100 microM HC-3. After a 3 h treatment, the cytosolic CTP:cholinephosphate cytidylyltransferase (CT) was activated by 56 per cent, while choline kinase (CK) was inhibited slightly. The stimulation of CT was not simply due to the intact HC-3 molecule, and there was no redistribution of CT between cytosol and microsomes. Taken together, the results suggest that HC-3 activates PC biosynthesis apart from the inhibitory effect on choline uptake.