EGF receptor down-regulation attenuates ligand-induced second messenger formation

Epidermal growth factor (EGF)-induced increases in cytosolic Ca2+ and inositol polyphosphate production were compared in a human hepatocellular carcinoma-derived cell line, PLC/PRF/5, and in an EGF receptor-overexpressing subline, NPLC/PRF/5. Formation of these second messengers was correlated to EGF receptor display at the cell surface by monitoring ligand-induced EGF receptor down-regulation. Both cell lines exhibited a strikingly similar cytosolic Ca2+ increase upon exposure to EGF. The initial inositol phosphate responses were also similar in the two cell lines; inositol 1,4,5-trisphosphate increased within 10-15 s and returned to prestimulatory values after 2 min in both cell lines, while inositol tetrakisphosphate and inositol 1,3,4-trisphosphate were elevated after a 2-min exposure to EGF. At later times the responses were markedly different; NPLC/PRF/5 cells exhibited prolonged production of inositol 1,3,4-trisphosphate and inositol tetrakisphosphate (maximum at 1-3 h) but PLC/PRF/5 cells showed decreased levels of these isomers after 10 min and a return to basal values by 1 h. Exposure of PLC/PRF/5 cells to EGF caused a progressive decrease in the amount of EGF receptor at the cell surface whereas such treatment did not change the surface receptor levels in NPLC/PRF/5 cells. Kinetic analysis of EGF receptor down-regulation showed that receptor internalization was rapid enough to account for the transient nature of the inositol phosphate response in PLC/PRF/5 cells. Thus, the divergent patterns of signaling exhibited by the two cell lines may reflect differences in the efficiency of EGF-induced down-regulation of surface receptors.     

Elucidation of an A and L system for amino acid transport in the human lymphoblast using a membrane filtration technique.

Optimum conditions have been established for the measurement of amino acid transport by human lymphoblastoid cell lines using a membrane-filtration technique. The parameters we found to be important for the reproducibility of the method are: the types and combination of filters, the strength of the vacuum applied to the filters and the density of the cultures at the time of harvesting and during uptake and filtration. We found that bovine serum albumin added to phosphate buffered saline (PBS) glucose in which the cells are washed, resuspended and assayed is essential for the maintenance of viability, the prevention of clumping and the retention of the accumulated amino acid. Using this procedure we have characterized two transport systems for the neutral amino acids; an A and an L system, which are similar but not identical to the A and L systems characterized in rodent cell lines. These A and L systems have characteristically lower Km's and Vm's for alanine and phenylalanine, when compared to rodent cell lines. In addition, we find alpha-AIB to be a poor competitor of alanine and phenylalanine uptake.     

Species-specific differences in the toxicity of puromycin towards cultured human and Chinese hamster cells

The toxicity of the protein synthesis inhibitor puromycin towards a number of human and Chinese hamster cell lines has been examined. In comparison to cells of human origin, Chinese hamster cells exhibited about 25-fold higher resistance towards puromycin. These differences appeared to be species related as all the cell lines from any one species showed similar sensitivity towards puromycin. The incorporation of [3H]leucine in the hamster cell lines was accordingly found to be more resistant to the inhibitory effects of puromycin as compared to human cells. Studies on the cellular uptake of [3H]puromycin showed that in comparison to human cells, the drug uptake/binding in the hamster cell lines was greatly reduced. However, protein synthesis in the extracts of hamster and human cells showed no significant differences in sensitivity towards puromycin. These results show that the observed species related differences in cellular toxicity to puromycin are due to differences in the cellular uptake/binding of the drug.     

Effect of Membrane Polyunsaturation on Carrier-Mediated Transport in Cultured Retinoblastoma Cells: Alterations in Taurine Uptake

Neural cell membranes naturally contain a large amount of polyunsaturated fatty acid, but the functional significance of this is unknown. An increase in membrane polyunsaturation has been shown previously to affect the high-affinity transport systems for choline and glycine in cultured human Y79 retinoblastoma cells. To test the generality of membrane polyunsaturation effects on transport, we investigated the uptake of other putative neurotransmitters and amino acids by these cells. Taurine, glutamate, and leucine were taken up by both high- and low-affinity transport systems, whereas serine, gamma-aminobutyrate, and alpha-aminoisobutyrate were taken up only by low-affinity systems. The high-affinity taurine and glutamate and low-affinity serine uptake systems were Na+ dependent. Arachidonic acid (20:4) supplementation of Y79 cells produced enrichment of all the major microsomal phosphoglycerides with 20:4, while docosahexaenoic acid (22:6) supplementation produced large increases in the 22:6 content of all fractions except the inositol phosphoglycerides. Enrichment with these polyunsaturated fatty acids facilitated taurine uptake by lowering the K'm of its high-affinity transport system. By contrast, enrichment with oleic acid did not affect taurine uptake. Glutamate, leucine, serine, gamma-aminobutyrate, and alpha-aminoisobutyrate uptake were not affected when the cells were enriched with any of these fatty acids. These findings demonstrate that only certain transport systems are sensitive to the polyunsaturated fatty acid content of the retinoblastoma cell membrane. The various transport systems either respond differently to changes in membrane lipid unsaturation, or they are located in lipid domains that are modified to different extents by changes in unsaturation.     

Elucidation of an a and L system for amino acid transport in the human lymphoblast using a membrane filtration technique

Summary Optimum conditions have been established for the measurement of amino acid transport by human lymphoblastoid cell lines using a membrane-filtration technique. The parameters we found to be important for the reproducibility of the method are: the types and combination of filters, the strength of the vacuum applied to the filters and the density of the cultures at the time of harvesting and during uptake and filtration. We found that bovine serum albumin added to phosphate buffered saline (PBS) glucose in which the cells are washed, resuspended and assayed is essential for the maintenance of viability, the prevention of clumping and the retention of the accumulated amino acid. Using this procedure we have characterized two transport systems for the neutral amino acids; an A and an L system, which are similar but not identical to the A and L systems characterized in rodent cell lines. These A and L systems have characteristically lower Km's and Vm's for alanine and phenylalanine, when compared to rodent cell lines. In addition, we find α-AIB to be a poor competitor of alanine and phenylalanine uptake. This work was supported by Grant No. CA18644, awarded by the National Cancer Institute, Department of Health, Education and Welfare, and from a grant from the National Science Foundation under Grant No. PCM 76-24328.     

Retinoic acid treatment of fibroblasts causes a rapid decrease in [3H]inositol uptake

NIH 3T3 fibroblasts treated with all-trans-retinoic acid (RA) showed a dramatic decrease in the uptake of [3H]inositol compared to solvent-treated controls. The onset of RA-induced inhibition of [3H]inositol uptake was rapid with a 10-15% decrease occurring after 2-3 h of RA exposure and 60-70% reduction after 16 h of RA treatment. A progressive dose-dependent decrease in inositol uptake was found as the concentration of RA increased from 10(-8) to 10(-5) M and the effect was fully reversible within 48 h after RA removal. The Vmax and Kt for the controls were 10 nmol/2.5 x 10(6) cells/2 h and 51 microM; and for RA-treated cells the values were 4 nmol/2.5 x 10(6) cells/2 h and 52 microM. The decreased [3H]inositol uptake was not due to a change in the affinity (Kt) of the transporter for the inositol but to a decrease in the Vmax. The maximal effect on inositol uptake was dependent on RA treatment of the cells after they reached saturation density or if made quiescent by serum starvation. RA was the most active of the different retinoids examined in the order RA greater than 13-cis-RA = retinyl acetate greater than all-trans-retinol greater than 5,6-dihydroxyretinoic acid methyl ester greater than N-4-hydroxyphenyl retinamide. In contrast to this effect on inositol, the uptake of fucose, mannose, galactose, and glucose was either not affected or enhanced (for mannose and fucose) by RA treatment. RA inhibition of inositol uptake was also observed in 3T3-Swiss and Balb/3T3 cells but not in two virally transformed 3T3 cell lines. Phlorizin, amiloride, and monensin inhibited inositol uptake by 66, 74, and 58%, respectively, and this inhibition was additive when the cells were treated with RA as well as these inhibitors. A decreased incorporation of [3H]inositol into polyphosphoinositides was also observed in RA-treated cells but not to the same extent as for [3H]inositol uptake. In conclusion, RA treatment of 3T3 fibroblasts decreases the uptake of [3H]inositol by up to 70% within 8 to 10 h at near physiological concentrations in a reversible and specific manner.     

Changes in inositol transport during DMSO-induced differentiation of HL60 cells towards neutrophils

[3H]Inositol uptake by HL60 cells was measured during DMSO-induced differentiation towards neutrophils. The values for Km (53.2 microM) and Vmax (5.3 pmol/min per 10(6) cells) obtained for control HL60 cells are in good agreement with previously published figures for this cell line. Inositol transport into HL60 cells was an active, saturable and specific process which was unaffected by extracellular glucose concentrations. Inositol transport rates changed during DMSO-induced differentiation of HL60 cells towards neutrophils. An increase in inositol transport rates occurred during the first 4 days of exposure to 0.9% DMSO and was concommitant with the period leading to growth arrest and prior to the acquisition of the differentiated phenotype. These changes preceded the rise in intracellular inositol concentration from 10.9 to 132.7 microM seen between day 1 and day 5. After 4 days exposure to DMSO the rate of inositol transport fell to a value of 3.2 +/- 0.3 pmol/min per 10(6) cells at day 7, this was accompanied by a small reduction in intracellular inositol from a peak value of 132.7 to 112 microM. The inositol transport rate, thus, appears to closely accompany changes in the intracellular concentration of inositol. Inositol transport in human peripheral blood neutrophils was an order of magnitude slower than the value for uninduced HL60 cells, but the Km for inositol transport was similar in both cell types and was unchanged during HL60 differentiation. This suggests that changes in inositol transport rate are achieved by the modulation of a commonly expressed inositol transporter, one consequence of which is the alteration of intracellular inositol concentrations.     

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.     

Differences in the metabolism of inositol and phosphoinositides by cultured cells of neuronal and glial origin

Phosphoinositide and inositol metabolism was compared in glioma (C6), neuroblastoma (N1E-115) and neuroblastoma X glioma hybrid (NG 108-15) cells. All cell lines had similar proportions of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2). Neuroblastoma and hybrid cells had almost identical phospholipid and phosphoinositide compositions and similar activities for the enzymes metabolizing polyphosphoinositides (PI kinase, PIP phosphatase, PIP kinase, PIP2 phosphatase, PIP2 phosphodiesterase). Glioma cells differed by having greater proportions of ethanolamine plasmalogen and sphingomyelin, lower PIP kinase, 3-5-fold higher PIP phosphatase activity and 10-15-fold greater PIP2 phosphodiesterase activity. Higher PIP phosphatase and PIP2 diesterase activities appear to be characteristic of cells of glial origin, since similar activities were found in primary cultures of astroglia. Glioma cells also metabolize inositol differently. In pulse and pulse-chase experiments, glioma cells transported inositol into a much larger water-soluble intracellular pool and maintained a concentration gradient 30-times greater than neuroblastoma cells. Label in intracellular inositol was less than in phosphoinositides in neuroblastoma and exchanged rapidly with extracellular inositol. In glioma, labeling of intracellular inositol greatly exceeded that of phosphoinositides. As a consequence, radioactivity in prelabeled phosphoinositides could not be effectively chased from glioma cells by excess unlabeled inositol. Such differences between cells of neuronal and glial origin suggest different and possibly supportive roles for these two cell types in maintaining functions regulated through phosphoinositide-linked signalling systems in the central nervous system.     

Differentiated human NT2-N neurons possess a high intracellular content of myo-inositol

myo-Inositol plays a key role in signal transduction and osmotic regulation events in the CNS. Despite the known high concentrations of inositol in the human CNS, relatively little is known about its distribution within the different cell types. In this report, inositol homeostasis was studied in NT2-N cells, a unique cell culture model of human CNS neurons. Differentiation of precursor NT2 teratocarcinoma cells into NT2-N neurons by means of retinoic acid treatment resulted in an increase in inositol concentration from 24 to 195 nmol/mg of protein. After measurement of intracellular water spaces, inositol concentrations of 1.6 and 17.4 mM were calculated for NT2 and NT2-N cells, respectively. The high concentrations of inositol in NT2-N neurons could be explained by (1) an increased uptake of inositol (3.7 vs. 1.6 nmol/mg of protein/h, for NT2-N and NT2 cells, respectively) and (2) a decreased efflux of inositol (1.7%/h for NT2-N neurons vs. 9.0%/h for NT2 cells). Activity of inositol synthase, which mediates de novo synthesis of inositol, was not detected in either cell type. The observation that CNS neurons maintain a high intracellular concentration of inositol may be relevant to the regulation of both phosphoinositide signaling and osmotic stress events in the CNS.     

Scavenger receptor class B,type I on non-malignant and malignant human epithelial cells mediates cholesteryl ester-uptake from high density lipoproteins

Hepatoma cell lines serve as a suitable model to study hepatic clearance of lipoprotein-associated cholesteryl esters (CEs). The present study aimed at investigating holoparticle-association of and selective CE-uptake from human high density lipoprotein subclass 3 (HDL3) by non-malignant adult (Chang-liver) and non-malignant fetal (WRL-68) epithelial cell lines as well as a hepatocellular carcinoma (HUH-7) cell line. Binding properties of 125I-HDL3 at 4 and 37 degrees C were similar for all three cell lines while degradation rates were highest for Chang-liver cells. Calculating the selective uptake of HDL3-associated CEs as the difference between [3H]CE- and 125I-HDL3 cell-association revealed that the selective lipid uptake and holoparticle-association was similar in Chang-liver while in WRL-68 and HUH-7 cells pronounced capacity for lipid tracer uptake in excess of holoparticle uptake was measured. Using RT-PCR, Northern and Western blot analysis, as well as immunocytochemical technique pronounced expression of scavenger receptor class B, type I (SR-BI) but not SR-BII (a splice variant of SR-BI less efficient for selective CE-uptake than SR-BI) could be identified in HUH-7 and WRL-68 cells. A polyclonal antiserum raised against SR-BI significantly decreased cell-association of [3H]CE-HDL3 in HUH-7 and WRL-68. The present findings suggest that the capacity for selective cholesteryl ester-uptake from high density lipoprotein by malignant and normal epithelial cells from the liver depends on expression of the scavenger receptor class B, type I.     

A high level of cell surface phosphatidylinositol-specific phospholipase C activity is characteristic of growth-arrested 3T3 fibroblasts but not of transformed variants.

Confluent monolayers of four contact-inhibited mouse fibroblast lines (Swiss 3T3, Balb/c 3T3, NIH 3T3, and C3H10T1/2) were found to have substantial levels of a cell surface phosphatidylinositol-specific phospholipase C (ecto-PLC). In contrast, confluent cultures of virally, chemically, or spontaneously transformed variants derived from these cell lines expressed undetectable or negligible levels of this enzyme activity. A simple and rapid assay, using lysophosphatidylinositol radio-labeled in the inositol group ([3H]-lysoPI) as the substrate was developed to provide a quantitative measure of the phospholipase C activity present at the external cell surface. For cells testing positive for ecto-PLC activity, rapid uptake of [3H]-lysoPI is accompanied by the simultaneous appearance of [3H]-inositol phosphate in the external medium. Confluent monolayers of the four mouse fibroblast lines exhibiting density-dependent growth inhibition had levels of ecto-PLC activity in the range of 50-800 pmol/min/10(6) cells (i.e., about 20-50 times greater than the activity observed for the transformed variants). The expression of ecto-PLC activity at the cell surface of the Swiss or Balb/c cells was dependent on the state of cell proliferation. Cultures which had become quiescent through attainment of confluence displayed a tenfold increased activity over that of subconfluent, growing cultures of these cells. Similarly, subconfluent Swiss 3T3 cells which had become quiescent following exposure to low serum conditions also showed increased activity. These results indicate that there may exist a correlation between the control of cell proliferation in contact-inhibited mouse fibroblasts and the expression of inositol phospholipid-specific phospholipase C activity at the external cell surface.     

Serum-free transfection of CHO cells with chemically defined transfection systems and investigation of their potential for transient and stable transfection

The generation of transgenic cell lines is acquired by facilitating the uptake and integration of DNA. Unfortunately, most of the systems generating stable expression systems are cost and time-consuming and transient expression is optimized to generate milligram amounts of the recombinant protein. Therefore we improved and compared two transfection systems, one based on cationic liposomes consisting of DOTAP/DOPE and the second one on polyethylenimine (PEI). Both systems have been used as chemically defined transfection systems in combination with serum-free cultivated host cell line. At first we had determined the toxicity and ideal ratio of DNA to PEI followed by determination of the optimal transfection conditions in order to achieve maximum transfection efficiency. We then directly compared DOTAP/DOPE and PEI in transient transfection experiments using enhanced green fluorescence protein (EGFP) and a human monoclonal antibody, mAb 2F5, as a model protein. The results which were achieved in case of EGFP were more than 15% transfectants at a viability of 85%. Despite the fact that expression of the mAb was found negligible we used both techniques to generate stable mAb 2F5 expressing cell lines that underwent several cycles of screening and amplification with methotrexate, and resulted in cell lines with similar volumetric production titers. These experiments serve to demonstrate the potential of stable cell lines even in case where the transient systems did not show satisfying results.     

Effects of bombesin on human small cell lung cancer cells: evidence for a subset of bombesin non-responsive cell lines

The effects of bombesin on three human small cell lung carcinoma cell (SCLC) lines (NCI-H69, NCI-H128, and NCI-H345) have been examined and compared to the effects of the peptide on the mouse fibroblast cell line Swiss 3T3, and the rat pituitary tumor cell line GH3W5. While all three SCLC lines expressed messenger RNA encoding pro-gastrin releasing peptide (GRP), only the NCI-H345 cells expressed detectable membrane receptors for GRP and responded to nanomolar concentrations of bombesin as shown by 125I-GRP binding, total inositol phosphate accumulation, and increased clonal growth in soft agarose. These data show that some SCLC lines are insensitive to bombesin and do not express detectable membrane receptors for GRP.     

The H(+)-dependent reduced folate carrier 1 of humans and the sodium-dependent methotrexate carrier-1 of the rat are orthologs

Previously, two different carrier systems for uptake of reduced folates and the antifolate methotrexate (Mtx) were described: the pH-dependent folate sensitive reduced folate carrier 1 (RFC1) from human, hamster and mouse and a sodium-dependent and folate insensitive Mtx carrier-1 (MTX-1) from rat. It was found that all critical residues of the homologous amino acid sequence were identical. RFC1- as well as MTX-1-mediated uptake of a marker substrate into suitable human and rat cell lines increased with proton concentration, was sodium-dependent at neutral pH, and inhibited by folate at acidic pH. It is concluded that RFC1 and MTX-1 are orthologs.     

L-proline uptake in human fibroblasts: evidence for a high-affinity system in addition to system A

Proline uptake was studied in human skin fibroblasts by simultaneous running of kinetic and inhibition experiments on the same cell lines. Two systems for proline uptake were shown: a high-affinity system not inhibited by alpha-(methylamino)isobutyric acid and a low affinity system inhibited by this amino acid (i.e. system A). These results appear to be of interest, firstly because up till now, system A was considered preferable for proline uptake in human fibroblasts, and secondly because they illustrate the need for combined inhibition and kinetic studies of amino acid uptake, especially when the substrate concentration range used and the respective Km of the systems do not allow their detection by kinetic analysis alone. Furthermore, this high-affinity system may have major physiological implications.     

Expression and functional features of NaCT, a sodium-coupled citrate transporter, in human and rat livers and cell lines

In this article, we report on the expression and function of a Na(+)-coupled transporter for citrate, NaCT, in human and rat liver cell lines and in primary hepatocytes from the rat liver. We also describe the polarized expression of this transporter in human and rat livers. Citrate uptake in human liver cell lines HepG2 and Huh-7 was obligatorily dependent on Na+. The uptake system showed a preference for citrate over other intermediates of the citric acid cycle and exhibited a Michaelis constant of approximately 6 mM for citrate. The transport activity was stimulated by Li+, and the activation was associated with a marked increase in substrate affinity. Citrate uptake in rat liver cell line MH1C1 was also Na+ dependent and showed a preference for citrate. The Michaelis constant for citrate was approximately 10 microM. The transport activity was inhibited by Li+. Primary hepatocytes from the rat liver also showed robust activity for Na+-coupled citrate uptake, with functional features similar to those described in the rat liver cell line. Immunolabeling with a specific anti-NaCT antibody showed exclusive expression of the transporter in the sinusoidal membrane of hepatocytes in human and rat livers. This constitutes the first report on the expression and function of NaCT in liver cells.     

Characterization of a Chinese hamster-human hybrid cell line with increased system L amino acid transport activity.

We have studied leucine transport in several Chinese hamster-human hybrid cell lines obtained by fusion of a temperature-sensitive line of Chinese hamster ovary cells, ts025C1, and normal human leukocytes. A hybrid cell line exhibiting a twofold increase in L-leucine uptake over that in the parental cell line was found. This hybrid cell line, 158CnpT-1, was temperature resistant, whereas the parental Chinese hamster ovary mutant, ts025C1, contained a temperature-sensitive leucyl-tRNA synthetase mutation. An examination of the different amino acid transport systems in this hybrid cell line revealed a specific increase of system L activity with no significant changes in systems A and ASC. The Vmax for L-leucine uptake exhibited by the hybrid 158CnpT-1 was twice that in the CHO parental mutant, ts025C1. Cytogenetic analysis showed that the hybrid 158CnpT-1 contains four complete human chromosomes (numbers 4, 5, 10, and 21) and three interspecific chromosomal translocations in a total complement of 34 chromosomes. Biochemical and cytogenetic analysis of segregant clones obtained from hybrid 158CnpT-1 showed that the primary temperature resistance and high system L transport phenotypes can be segregated from this hybrid independently. The loss of the primary temperature resistance was associated with the loss of the human chromosome 5, as previously reported by other laboratories, whereas the loss of the high leucine transport phenotype, which is associated with a lesser degree of temperature resistance, was correlated with the loss of human chromosome 20.     

Thrombin's effects on osteoblastic cells. I. Cytosolic calcium and phosphoinositides

Thrombin, a blood coagulation factor, has been shown to be a very effective in vitro bone resorbing agent whose mechanism of action on osteoblastic cells remains to be elucidated. In the present study, the effects of highly purified human thrombin on Saos-2 and G292 cells, two human osteoblast-like osteosarcoma cell lines, were investigated. Thrombin (0.6-16 U/ml) caused a significant, dose-dependent increase in osteoblastic cell proliferation. Thrombin also elicited a dose-dependent increase in cytosolic calcium concentration in both Saos-2 and G292 cells (maximal increases were 38% and 200% over baseline, respectively). Addition of thrombin to the osteoblast-like cells resulted in significant time- and dose-dependent changes in phosphoinositide levels: the percentage of inositol monophosphate levels were decreased, whereas the percentage of inositol bisphosphate, inositol trisphosphate and inositol tetrakisphosphate levels were increased. The relative magnitude of the changes in phosphoinositide levels was similar to the changes in cytosolic calcium concentration. These results suggest that thrombin's mechanism of action on bone cells may involve increases in cytosolic calcium levels and in phosphoinositide metabolism.