Structure–activity relationships in the conversion of vitamin K analogues into menaquinone-4. Substrates essential to the synthesis of menaquinone-4 in cultured human cell lines

To reveal an essential biological role of menaquinone-4, we have clarified that dietary PK was converted to menaquinone-4 (MK-4) in animal tissues using deuterated vitamin K analogues. However, the kinds of analogue converted into MK-4 have not been elucidated. In this study, we examined structure–activity relationships in the conversion of several vitamin K analogues, with a substituted side chain, into MK-4 using cultured human cell lines. The results differed with the side chain of the analogues, that is, (1) the length of the isoprene unit and (2) the number of double bonds in the side chain. These findings would be useful for clarifying the mechanism of conversion of other vitamin K homologs into MK-4 as well as related enzymes.     

Myoinositol uptake by four cultured mammalian cell lines

The uptake of myo-[2-3H]inositol by mouse neuroblastoma, human Y79 retinoblastoma, human HL60, and bovine pulmonary artery endothelial cells occurs by a saturable, Na+-dependent and partially energy-dependent mechanism. Inositol uptake by all four cell lines occurred by both a high-and low-affinity system. The kinetic parameters for the high-affinity uptake systems were similar for all four cell lines. These data suggest that all four of these diverse cell lines have similar inositol transport systems and probably rely on extracellular inositol for anabolic processes.     

Methionine utilization in long-term human lymphoid cell lines

Long-term lymphoid cell lines (LTL) cultured under normal conditions use methionine primarily for protein synthesis, although a significant proportion is converted to S-adenosylmethionine (SAM) and used for synthesis of the polyamines, spermidine, and spermine. When LTL are cultured under conditions of high cell density, there is an initial phase of rapid protein synthesis and accumulation of SAM as found under normal culture conditions, but this soon ceases. Polyamine synthesis is small under these conditions, despite the presence of relatively large amounts of SAM.     

Thymosin β4 in cultured mammalian cell lines

Thymosin β₄, originally isolated from calf thymus [Low et al., Proc. Nat. Acad. Sci. USA78, 1162–1166 (1981)] is present in a number of cell lines unrelated to the reticuloendothelium, including myoblasts and fibroblasts. It is also actively synthesized by these cell lines. Its content and rate of synthesis in the cell lines examined appear to be correlated with their ability to adhere and their motility.     

Conversion of phylloquinone (Vitamin K1) into menaquinone-4 (Vitamin K2) in mice: two possible routes for menaquinone-4 accumulation in cerebra of mice

There are two forms of naturally occurring vitamin K, phylloquinone and the menaquinones. Phylloquinone (vitamin K(1)) is a major type (>90%) of dietary vitamin K, but its concentrations in animal tissues are remarkably low compared with those of the menaquinones, especially menaquinone-4 (vitamin K(2)), the major form (>90%) of vitamin K in tissues. Despite this great difference, the origin of tissue menaquinone-4 has yet to be exclusively defined. It is postulated that phylloquinone is converted into menaquinone-4 and accumulates in extrahepatic tissues. To clarify this, phylloquinone with a deuterium-labeled 2-methyl-1,4-naphthoquinone ring was given orally to mice, and cerebra were collected for D NMR and liquid chromatography-tandem mass spectrometry analyses. We identified the labeled menaquinone-4 that was converted from the given phylloquinone, and this conversion occurred following an oral or enteral administration, but not parenteral or intracerebroventricular administration. By the oral route, the phylloquinone with the deuterium-labeled side chain in addition to the labeled 2-methyl-1,4-naphthoquinone was clearly converted into a labeled menaquinone-4 with a non-deuterium-labeled side chain, implying that phylloquinone was converted into menaquinone-4 via integral side-chain removal. The conversion also occurred in cerebral slice cultures and primary cultures. Deuterium-labeled menadione was consistently converted into the labeled menaquinone-4 with all of the administration routes and the culture conditions tested. Our results suggest that cerebral menaquinone-4 originates from phylloquinone intake and that there are two routes of accumulation, one is the release of menadione from phylloquinone in the intestine followed by the prenylation of menadione into menaquinone-4 in tissues, and another is cleavage and prenylation within the cerebrum.     

End products of glucose and glutamine metabolism by cultured cell lines

Rates of CO2 production from glucose and glutamine, intracellular metabolite levels, and release of metabolic end products into the culture medium were determined for 13 cultured cell lines, including a glycolysis-defective mutant. All the non-mutant lines synthesized pyruvate, lactate, alanine, proline, aspartate, and citrate, so that the metabolism of glucose and glutamine resulted mainly in the production of these compounds and only to a lesser extent in complete oxidation to CO2. These data and the pattern of metabolites produced by the mutant line were consistent with a model characterized by incomplete glutamine oxidation leading to end product accumulation. Multiple linear regression analysis identified the metabolite levels most highly correlated with the intracellular citrate level and with the amount of citrate released into the medium. The analysis also showed that the rates of CO2 production from glucose and glutamine were themselves positively correlated, suggesting that the oxidation of the two substrates is coordinately controlled under normal culture conditions.     

Regulation of retinol-binding protein metabolism in cultured rat liver cell lines

Retinol-binding protein (RBP), the plasma transport protein for vitamin A, is synthesized and secreted by the liver. In vitamin A deficiency, RBP secretion is blocked, leading to low serum and high liver levels of RBP. Administration of retinol to the intact rat stimulates a rapid secretion of RBP from liver into serum. We explored the use of a liver cell culture system to study the regulation of the synthesis and secretion of RBP. We found two lines of differentiated rat hepatoma cells, MH₁C₁ and H₄ II EC₃ (H₄), that synthesized RBP during culture in vitro. The net synthesis of RBP was a function of the number of cells per dish and the duration of incubation. Both cell lines synthesized RBP when incubated in Neuman and Tytell's Serumless Medium (NTS medium), while the MH₁C₁ cells also synthesized RBP in Ham's F-12 medium with added serum. A relatively large proportion (14–56%) of the RBP was retained within the cells when they were incubated in the vitamin A-free NTS medium alone. Addition of serum to NTS medium stimulated the release of RBP from the cells into the medium and also increased the net synthesis of RBP. These effects were not due to the increased adhesion of the cells to the petri dish. Addition of retinol (at levels of 0.35 or 3.5 nmole/ml) to the NTS medium resulted in the stimulation of RBP secretion from the cells into the medium and an increase in the net synthesis of RBP. By contrast, retinol had no effect on either the net synthesis or the cell-to-medium distribution of rat serum albumin. The data from these cell lines in culture suggest that retinol has a specific regulatory effect on RBP metabolism. These cells thus resemble the normal rat liver cell in vivo in regard to the known regulation of RBP metabolism.     

Steroid metabolism in human breast cancer cell lines

The metabolism of 1,2-³H-androstenedione was studied in 2 cell lines, MCF-7 (estrogen responsive) and BT-20 (estrogen nonresponsive) over 48 hrs. Water soluble and unconjugated metabolites were separated by solvent partition and the former was submitted to chromatography on Sephadex LH-20 and enzyme hydrolysis. The resulting unconjugated steroids were separated by paper chromatography and identities were established by reverse isotope dilution. The unconjugated steroids initially obtained were separated by chromatography and identified by reverse isotope dilution. About 70% of the androstenedione was metabolized by both cell lines. However, the respective conversions to conjugates by MCF-7 and BT-20 were 31% and 0.32%. In the former, glucosiduronates predominated (94%) and consisted of androsterone (55%), etiocholanolone (9.4%) and androstanediol (5α-androstane-3α,17β-diol) (9.3%). Androsterone comprised most of the unconjugated metabolites in both cell lines. Androstanediol was found in both cell lines, 2% in MCF-7 and 12% in BT-20. Testosterone, 5α-androstane-3,17-dione and 3β-hydroxy-5α-androstan-17-one were isolated only from MCF-7. The metabolism of ³H-estriol was studied in a similar way. Both cell lines produced about equal amounts of estriol-3-sulfate (9%) and a compound with properties of estriol-3-glucosiduronate (0.15 – 0.5%). The results worthy of emphasis are: 1. The far greater conjugation of androgens exhibited by the MCF-7 cell lines as compared to the BT-20 cell lines; 2. In MCF-7, the high conversion of androstenedione to etiocholanolone (glucosiduronate form), a metabolite reported to form only in liver and sebaceous cysts; 3. The possible formation in both cell lines of estriol-3-glucosiduronate, normally a metabolite of the intestine.     

Presence of human papillomavirus genome in human tumor cell lines and cultured cells.

A series of 47 human carcinoma cell lines and their cultured cells were examined for human papillomavirus (HPV) genomes with the use of an HPV detection kit (DNA-RNA hybridization, mixed HPV DNA probe of types 6, 11, 16, 18, 31, 33 and 35). Four of 8 cases of mild dysplasia, 3 of 9 cases of severe dysplasia, 3 of 7 cases of carcinoma in situ, 3 of 15 cases of uterine carcinoma and 5 of 6 cases of condyloma acuminatum were shown to contain the HPV DNA genome in primary cultured cells, while HPV was not detected in the third-passage cells except for the three cases of large cell, nonkeratinizing squamous cell carcinoma. HPV was also not detected in such normal tissues as uterine cervical squamous epithelium, uterine cervical columnar epithelium and endometrium. The presence of HPV DNA genomes was detected consistently in the passages of three lines (SKG-II, HKMUS and HKTUS; large cell nonkeratinizing squamous cell carcinomas of the uterine cervix) with the use of the Southern Blot method (DNA-DNA hybridization, mixed HPV probe of types 6, 11, 16 and 18). HPV type 16 DNA was detected in HKTUS, and HPV type 18 DNA was found in SKG-II and HKMUS. The other 44 cell lines, including ovarian carcinoma, endometrial carcinoma, sarcoma, gastric cancer, pancreatic cancer and rectal cancer, were negative for the HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33 and HPV-35 genomes under stringent hybridization conditions.     

Ca-dependent modulation of human glycine receptors expressed in cultured cell lines

Glycine receptors (GlyRs) provide the main inhibitory neurotransmission in spinal cord and brainstem synapses of vertebrates. Fucile et al. (2000) discovered that elevation of intracellular Ca2+ caused rapid potentiation of GlyRs. This modulation develops in less than 100 ms. It is characterized by an increase in GlyR apparent affinity for glycine. It has been suggested that the phenomenon of Ca-induced potentiation involves an unknown Ca2+-binding protein (CaBP). Using the yeast two-hybrid system, screening of human brain cDNA library against the cytoplasmic loop of human alpha 1 subunit (GlyRhl) allowed us to identify five new interactors. One of them belongs to a family of Ca-binding proteins. We analyzed effect of "short" forms of this protein (CaBP-S) on functional properties of GlyRhl expressed in HEK-293 and CHO cells. Using whole-cell recordings and rapid agonist application we constructed concentration dependencies of glycine-induced currents. This analysis revealed statistical differences in EC50s between control cells (expressing only GlyRhl) and those expressing CaBP-S. In HEK-293 cells recorded under conditions of low intracellular Ca concentration (BAPTA 20 mM in the recording pipette), EC50 for glycine in control cells and expressing GlyRhl + CaBP-S were, correspondently, 68+/-49 microM (n = 29) and 409 +/-421 microM (n = 60). In CHO cells EC50 were 54+/-43 microM (n = 25) and 123 +/-104 microM (n = 28). These differences were statistically not significant at recording with intracellular solution containing high Ca concentration (50 microM). In this case EC50 were correspondently 35+/-28 microM (n = 7) and 64 +/-38 microM (n = 7). These results suggest that CaBP-S causes decrease of GlyR sensitivity to agonist through interaction with cytoplasmic domain of GlyR.     

Iron metabolism of established human hematopoietic cell lines in vitro

Three malignant hematopoietic cell lines were used in studies on cellular iron metabolism. Our results show that iron-carrying transferrin became bound to specific dimeric cell surface receptors. Iron accumulated within the cell with time, whereas intact transferrin was released back to the medium. Chloroquine and NH₄Cl, known as pH-raising agents in vesicles of the lysosomal system, inhibited iron accumulation and transferrin binding in a dose-dependent manner. This suggests that the acid pH in endosomes leads to the cleavage of the iron-transferrin bonds. Transferrin degradation was not found, which leads us to suggest a process of ‘acid flushing’ for the dissociation of iron from transferrin without the involvement of endosome-lysosome fusion. Taken together, the data agree with the concept of receptor-mediated endocytosis, as described for many macromolecules. Iron was stored in ferritin in the cell types tested. Only a minor part (less than 15%) of the iron was bound in hemoglobin in the K-562 cell line. The relationship between iron stores and exogenously added iron in heme synthesis was investigated using a double labelling (⁵⁵Fe/⁵⁹Fe) technique. The results showed that exogenous iron was preferentially used before the iron stored in ferritin. The results are discussed in relation to various hypotheses on cellular iron uptake and transport.     

Glucose uptake and metabolism by cultured human skeletal muscle cells: rate-limiting steps

To use primary cultures of human skeletal muscle cells to establish defects in glucose metabolism that underlie clinical insulin resistance, it is necessary to define the rate-determining steps in glucose metabolism and to improve the insulin response attained in previous studies. We modified experimental conditions to achieve an insulin effect on 3-O-methylglucose transport that was more than twofold over basal. Glucose phosphorylation by hexokinase limits glucose metabolism in these cells, because the apparent Michaelis-Menten constant of coupled glucose transport and phosphorylation is intermediate between that of transport and that of the hexokinase and because rates of 2-deoxyglucose uptake and phosphorylation are less than those of glucose. The latter reflects a preference of hexokinase for glucose over 2-deoxyglucose. Cellular NAD(P)H autofluorescence, measured using two-photon excitation microscopy, is both sensitive to insulin and indicative of additional distal control steps in glucose metabolism. Whereas the predominant effect of insulin in human skeletal muscle cells is to enhance glucose transport, phosphorylation, and steps beyond, it also determines the overall rate of glucose metabolism.     

Fatty acid uptake by human hepatoma cell lines represents a carrier-mediated uptake process

Cellular influx kinetics of a representative long chain fatty acid, [3H]oleate, were examined in monolayer cultures of three different human hepatoma cell lines (Hep G2; PLC/PRF 5; Mz-Hep-1). The cultures were incubated with 173 microM [3H]oleate in the presence of various concentrations of albumin which served to modulate the unbound oleate concentration in the medium. For all [3H]oleate-albumin complexes incubated, it was shown that cellular uptake of [3H]oleate over the initial 30 s incubation period was maximal, linear and independent of intracellular fatty acid metabolism, representing cellular influx. With increasing unbound oleate concentrations in the medium cellular influx by all three cell lines revealed similar saturation kinetics with Km values of 112.6 +/- 14.5 nM and Vmax values of 7.19 +/- 0.32 nmol.min-1 per mg cell protein. When these hepatoma cell lines were pretreated with the IgG fraction of a monospecific antibody to the rat liver membrane fatty acid binding protein (MFABP), initial uptake of [3H]oleate was selectively inhibited compared to controls pretreated with the IgG fraction of the preimmune serum. Furthermore, immunoblot analysis with the monospecific antibody to the rat MFABP revealed reactivity with a single 40 kDa protein in the homogenates of all three cell lines. These data suggest that uptake of fatty acids by human hepatoma cells may be mediated by a specific membrane fatty acid binding protein.     

Adenosine uptake, transport, and metabolism in human erythrocytes

Using rapid kinetic techniques, we have determined the kinetics of zero-trans influx and equilibrium exchange of adenosine, and its uptake and in situ phosphorylation at 25 degrees C in human erythrocytes which were pretreated with 2'-deoxycoformycin to inhibit deamination of adenosine. Both the Km and Vmax for adenosine transport were about 300 times higher than those for the in situ phosphorylation of adenosine (Km about 0.2 microM), so that the first order rate constants for both processes were about the same. In contrast, the first order rate constant for adenosine deamination by untreated, intact cells was about 20% of that of adenosine transport or phosphorylation. These kinetic properties of the various steps, in combination with substrate inhibition of adenosine phosphorylation above 1 microM adenosine, assure that, at extracellular concentrations of physiological relevance (less than 1 microM), adenosine is very rapidly and efficiently salvaged by the erythrocytes and converted to ATP, whereas at extracellular concentrations of 10 microM or higher, practically all adenosine transported into the cells is deaminated. When the concentration of adenosine was 0.1 microM, a 10% (v/v) suspension of erythrocytes depleted the extracellular fluid of adenosine within 1 min of incubation at 25 degrees C.     

Regulation of fibronectin and laminin binding activity in cultured human lymphoblastic cell lines

The current study shows that a clonal derivative of the Jurkat cell line up-regulates both the avidity and density of the α 6/β1 receptor in response to phorbol 12-myristate 13-acetate (PMA). This derivative attaches to fibronectin and, to a lesser degree, laminin constitutively. Adhesion and spreading are dramatically up-regulated following treatment with PMA. The response on fibronectin peaks within 4 hours, is insensitive to cyclohexaminde, can be blocked by monoclonal antibodies (Mabs) to the β1 and α 5 subunits of the β1 family of integrins, and is not associated with increased expression of the α 5 or β1 epitopes at the cell surface. In contrast, the response on laminin is biphasic. The early phase parallels the response on fibronectin. The second phase peaks after 48–72 hours of treatment with PMA, is sensitive to cycloheximide, can be blocked by Mabs to the β1 and α 6 subunits, and is associated with increased expression of the α 6 epitope. Both the density independent and dependent responses to PMA in Jurkat cells are blocked by the protein kinase inhibitor staurosporine. The HSB-2, CEM, Molt-4, and HPB-ALL T-lymphoblastic cell lines also up-regulate attachment to fibronectin and laminin following treatment with PMA. All four lines constitutively attach to fibronectin and show rapid up-regulation of attachment following treatment with PMA. None of the lines attach to laminin prior to PMA treatment; however, specific adhesion developed after 4–120 hours of treatment. The most mature lines (Jurkat and HPB-ALL) up-regulated adhesion on laminin more rapidly than the less phenotypically mature lines (CEM, Molt-4, and HSB-2). In summary, clonal derivatives of the Jurkat cell line up-regulated attachment to laminin through protein kinase dependent increases in α /β1 receptor avidity and density. In addition, the expression of functional receptors for laminin is linked to developmental maturity in a series of T-lymphoblastic cell lines. © 1993 Wiley-Liss, Inc.     

Comparative characteristics of three human embryonic stem cell lines

Human embryonic stem (hES) cells have unique features including unlimited growth capacity, expression of specific markers, normal karyotypes and an ability to differentiate. Many investigators have tried to use hES cells for cell-based therapy, but there is little information about the properties of available hES cell lines. We compared the characteristics of three hES cell lines. The expression of SSEA-1, -3, -4, and APase, was examined by immunocytochemistry, and Oct-4 expression was analyzed by RT-PCR. Differentiation of the hES cells in vitro and in vivo led to the formation of embryoid bodies (EBs) or teratomas. We examined the expression of tissue-specific markers in the differentiated cells by semiquantitative RT-PCR, and the ability of each hES cell line to proliferate was measured by flow cytometry of DNA content and ELISA. The three hES cell lines were similar in morphology, marker expression, and teratoma formation. However there were significant differences (P < 0.05) between the differentiated cells formed by the different cell lines in levels of expression of tissue-specific markers such as renin, kallikrein, Glut-2, beta- and delta-globin, albumin, and alpha1-antitrypsin (alpha1-AT). The hES cell lines also differed in proliferative activity. Our observations should be useful in basic and clinical hES cell research.     

Study of carbohydrate metabolism in glycogen storing cell lines derived from cultured rat hepatocytes

Some aspects of carbohydrate metabolism were investigated in three non-malignant, glycogen storing, cell lines derived from a primary culture of rat hepatocytes, and in the Morris hepatoma 3924 cells. The three cell lines show biochemical alterations which are, to a large extent, similar to those found in the hepatoma cells: increased activity of glycolytic enzymes and decreased activity of gluconeogenetic enzymes. An increase of glucose-6-phosphate dehydrogenase activity is also found. The three cell lines, as the Morris hepatoma cells, actively convert glucose into lactate under the in vitro conditions of culture. Fructose is not taken up as quickly as glucose and galactose is not metabolized. As compared with normal hepatocytes, the three cell lines have altered metabolism and growth behaviour. They largely resemble the preneoplastic cells appearing in rat liver at the early stages of experimental carcinogenesis.