Expression of cell cycle related proteins—proliferating cell nuclear antigen (PCNA) and statin—during adaptation and de-adaptation of EUE cells to a hypertonic medium

EUE cells adapted to grow for long times in a hypertonic medium have a longer cell cycle than those growing in isotonic medium. To elucidate whether this lengthening involves specific cycle phases to differing extents, the expression of two cycle-related protein, PCNA and statin, was studied by dual parameter flow cytometry of indirect immunofluorescence protein labelling and DNA content. In isotonic medium, most cells, in all the cycle phases, were PCNA positive; in contrast, PCNA negative cells and statin positive cells were very few in number and only fell in the G0/1 range of DNA contents. In hypertonic medium, the frequency of PCNA positive cells was lower, and that of statin positive cells higher, than in isotonic medium, particularly in the G0/1 range of DNA contents: this suggests that a G0 block occurs under long-term hypertonic stress. Consistently, dual parameter flow cytometric measurement of BrdUrd immunofluorescence labelling and DNA content showed that fewer cells entered S phase in hypertonic medium and their progression through the S phase was slower; evidence was also found for the occurrence of a G2 block. These kinetics changes were fully reversible in isotonic medium, thus indicating the adaptive nature of the EUE response to hypertonicity.     

Cell cycle effects of hypertonic stress on various human cells in culture

Long-term exposure to hypertonic (HT) culture media has been found to perturb the cell cycle and change gene expression in various animal cell types. A lower growth rate, with exit of cells from the cycling compartment has been observed previously in human transformed EUE. cells. The aim of this study was to investigate if the kinetic changes after long-term HT stress, were typical of transformed cells or could be also found in primary cultures of normal cells. Human transformed cells from normal and neoplastic tissues, and normal human cells of epithelial and connective origin have been studied. After the incorporation of bromodeoxyuridine (BrdUrd), the frequency of S-phase cells was estimated by dual-parameter flow cytometry of DNA content versus BrdUrd immuno-labelling; the total growth fraction was also estimated, after immunolabelling with an anti-PCNA antibody. We also investigated, by polyacrylamide gel electrophoresis, changes in the amount of a 35 kDa protien band, which increased in EUE cells grown in an HT medium, and which may be directly involved in cell resistance to hypertonicity. Lower BrdUrd labelling indices and higher frequencies of cells in the G_0/1 range of DNA content were common features of all the cells in HT media, irrespective of their tissue of origin; other cycle phases may also be involved, depending on the cell type considered. The mechanisms by which cells cope with the HT environment could however, differ, since only some cell types showed an increase of the 35 kDa stress protein found originally in HT EUE cells.     

A 33 kDa protein band is enhanced during long-term adaptation of EUE cells to a hypertonic medium

A cell line derived from human embryonic epithelium (EUE cells) shows an enhanced expression of a 33 kDa protein when adapted to grow in a hypertonic medium containing 0.246 M NaCl (1.8 x the isotonic concentration). The maximum amount of this protein, followed by SDS-PAGE electrophoresis, was found after 4 days of adaptation; thereafter, the protein band remained fairly constant up to 30 days. When the cells were transferred back to a medium containing 0.137 M NaCl (isotonic medium), the protein pattern reverted to that of control cells. This protein is mainly localized in the cytosol, although a small part is associated with the 150,000 g pellet and needs detergents to be extracted. The molecular weight and the cellular location suggest a possible analogy with the so-called amphitropic proteins, that are known to interact with both the epidermal growth factor receptor and hydrophobic structures, such as the membrane phospholipids and the cytoskeletal components.     

Effect of hypertonic medium on human cell growth: III. Changes in cell kinetics of EUE cells

The effects of hypertonicity on cell kinetics of EUE cells in culture have been investigated. After 4 days of growth in a hypertonic medium, the plating efficiency of EUE cells was reduced and cell growth was significantly slowed. Flow cytometric measurements of DNA content in synchronized cells, as well as flow cytometric determinations of DNA content and bromodeoxyuridine incorporation in asynchronous cells, also showed that the cell cycle is slowed in a hypertonic medium. In addition, the fraction of cycling cells is smaller and their progression through the S phase slower than in an isotonic medium.     

Effect of hypertonicity on survival of unprotected human cultured cells following freezing and thawing

An investigation was carried out on the post-thaw survival of unprotected human heteroploid EUE cells, either maintained in isotonic medium (0.137 M NaCl) or adapted to hypertonicity (0.356 M NaCl) and frozen in medium with an increased concentration of NaCl. A fivefold increase in the survival fraction of the adapted cells in comparison with the unadapted ones was observed when cells were frozen in isotonic medium. When cells were frozen in hypertonic medium (0.356 M NaCl), the two cell types exhibit comparable survival values. The results are discussed, with special attention to cell defense mechanisms against freezing injury.     

Effects of hypertonic and sodium-free medium on transport of a membrane glycoprotein along the secretory pathway in cultured mammalian cells

Incubation of cultured cells in hypertonic medium and sodium-free medium have been shown to block transport at two different stages along the endocytic pathway. To determine the effects of these treatments on the exocytic pathway, we studied the transport of the membrane glycoprotein of vesicular stomatitis virus (VSV-G) in cells infected with tsO45 mutant virus. This mutant synthesizes a VSV-G that accumulates in the endoplasmic reticulum (ER) when cells are incubated at 39.5 degrees C. In addition, VSV-G accumulates in the post-ER pre-Golgi compartment when cells are incubated at 15 degrees C and in the trans-Golgi network (TGN) when cells are incubated at 18 degrees C. Upon transfer of cells to 32 degrees C in control medium, VSV-G exits each of these compartments and is transported to the cell surface. Incubation in sodium-free medium at 32 degrees C did not block transport from any of these three compartments. In contrast, incubation in hypertonic medium blocked export from the ER, transport from the pre-Golgi compartment to the Golgi complex, and transport from the TGN to the cell surface. Our results, in combination with previous studies, suggest that hypertonic medium blocks at least five distinct transport steps; the three exocytic steps described here, endocytosis from the cell surface, and transport of cell surface proteins into the Golgi complex. This raises the possibility that vesicular transport in different parts of the cell shares common elements that are inhibited by this treatment.     

Changes in the ultrastructure of the chloride cells of sturgeon during adaptation to a hypertonic medium]

Alterations in the ultrastructure of chloride cells of young sturgeons Acipenser guldenstadti Brandt in the process of adaptation to hypertonic medium (S=105%) were under study. In the process of this adaptation thread-shaped long mitochondria became shorter, their random position was substituted by orientation in parallel to the axis of the cell. The amount of mitochrondria increased. A great number of vacuoles appeared in the cytoplasm. The character of the endoplasmic reticulum changed. The obtained data on the ultrastructure of chloride cells of the sturgeon confirm and supplement earlier light microscopy investigations of chloride cells of the sturgeon. The changes of the ultrastructure of chloride cells are interpreted as functional and adaptational ones.     

Modulation by phorbol 12-myristate 13-acetate of arachidonic acid release from rat basophilic leukemia cells stimulated with A23187

Rat basophilic leukemia (RBL-2H3) cells were cultured in medium containing [3H]arachidonic acid and labelling of the different lipid fractions was followed with time. After up to 4 h of culture, the label was found mostly in phosphatidylcholine. After 8 h, labelling of phosphatidylethanolamine gradually exceeded that of phosphatidylcholine, until at 24 h, approximate equilibrium labelling of the lipid fractions was attained and 45% of the label was found in phosphatidylethanolamine, 35% in phosphatidylcholine, 18% in the phosphatidylserine/inositide fraction and the remainder in the neutral lipid fraction. Stimulation of cells with A23187 after 30 min of labelling caused release of [3H]arachidonic acid which was accountable by a decrease in radioactivity of phosphatidylcholine, whereas stimulation of cells after 24 h of labelling caused the release of radioactive arachidonic acid, which was accompanied by a decrease of label in both phosphatidylcholine and phosphatidylethanolamine. Incubation of the labelled cells with phorbol 12-myristate 13-acetate prior to ionophore addition enhanced both the release of [3H]arachidonic acid and its metabolites and the decrease in label of the same phospholipids as those affected by ionophore alone. Under our conditions, the enhancement effects of phorbol ester were greatest after 2-5 min of preincubation, prior to ionophore addition. The results suggest that in basophilic leukemia cells, arachidonic acid release proceeds from several pools of phospholipids and that the activity of the phospholipase(s) involved is modulated by protein kinase C.     

Dynamics of Phospholipid Content in the Vacuolar Membrane of Red Beet Taproots Exposed to Abiotic Stress

The composition of vacuolar membrane phospholipids in the taproot of red beet (Beta vulgaris L.), cv. Modana, was determined at normal conditions and under different types of stress (hypo- and hyperosmotic and oxidative stress). The experiments have shown that, among vacuolar membrane phospholipids in red beet taproot, phosphatidylcholines and phosphatidylethanolamines dominated and accounted for 70% of total phospholipids. It is interesting that the content of phosphatidic acid was high (20% of total phospholipids of the vacuolar membrane). Stress effects brought about changes in the composition of membrane phospholipids, which may be an element of phenotypic adaptation. Under hypoosmotic stress, reliable changes in the content of phosphatidic acid were observed, hyperosmotic stress was associated with changes in the level of phosphatidylcholines and phosphatidylinositols, and oxidative stress was notable for changes in the content of phosphatidylethanolamines and phosphatidylserines. The most significant changes were observed in the classes of phospholipids that may be involved in structural modification of membranes associated with transformation of their bilayer lamellar structure into hexagonal. These phospholipids comprise phosphatidic acid, phosphatidylcholines, and phosphatidylethanolamines. Revealed changes in the content of these phospholipids may alter the ratio between lamellar bilayer and nonbilayer hexagonal lipid structures in the vacuolar membrane and act as an important adaptation mechanism ensuring protection against stress.     

An essential role of phosphatidylglycerol in the formation of the osmotically stable liposomes of Escherichia coli phospholipids

A temperature sensitive auxotroph of Escherichia coli K-12 requiring unsaturated fatty acids can grow normally at 28 degrees C, but requires an osmotic stabilizer such as a high amount of salt or sugar in the medium for the growth at 42 degrees C. Namely, the apparent osmotic stability of the cells at 28 degrees C and 42 degrees C is quite different. The osmotic properties of liposomes of the phospholipids extracted from these cells were investigated. The osmotically induced volume change of the multilamellar liposomes was examined by the turbidimetric method. The liposomes prepared from cells grown at 28 degrees C can swell and shrink under a wide range of hypo-and hypertonic conditions. However, those from cells grown at 42 degrees C could not swell under hypotonic conditions. These results exhibit a good correlation between the apparent osmotic stability of E. coli cells and the osmotic properties of the liposomes prepared from the extracted total phospholipids. To clarify the role of each phospholipid component, the osmotic properties of the liposomes reconstituted from the purified phospholipid species were further investigated. The results clearly showed that phosphatidylglycerol is the key factor that stabilizes the membranes of E. coli phospholipids against osmotic pressure.     

Inositol phospholipid metabolism in human platelets stimulated by ADP

ADP-induced changes in inositol phospholipids, phosphatidic acid and inositol phosphates of human platelets have been studied in detail, using not only 32P labelling, but also by examining changes in amounts of the phospholipids, their labelling with [3H]glycerol and their specific radioactivities; changes in the labelling of inositol phosphates in platelets prelabelled with [3H]inositol were also measured. During the early (10 s) stage of reversible ADP-induced primary aggregation in a medium containing fibrinogen and with a concentration of Ca2+ in the physiological range (2 mM), the amounts of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and phosphatidylinositol 4-phosphate (PtdInsP) decreased (by 11.2 +/- 4.9% and 11.3 +/- 5.3%, respectively) while the labelling, but not the amount, of phosphatidic acid increased. The decreases do not appear to be attributable to the action of phospholipase C because the specific radioactivity of phosphatidic acid labelling with [3H]glycerol was not significantly increased at 10 s (although the initial specific radioactivities of the inositol phospholipids and PtdCho were more than double that of phosphatidic acid), and no increases in the labelling of inositol trisphosphate (InsP3), inositol bisphosphate (InsP2) or inositol phosphate (InsP) were detectable at 10 s. Shifts in the interconversions between PtdInsP2 and PtdInsP, and PtdInsP and PtdIns may occur. By 30 to 60 s, when deaggregation was beginning, the amounts of PtdInsP2, PtdInsP and phosphatidic acid were not different from those in unstimulated platelets, but large increases in the 32P-labelling and [3H]glycerol labelling of phosphatidic acid were observed. Formation of [3H]inositol-labelled InsP3 was not detectable at any time in association with ADP-induced primary aggregation, indicating that degradation of PtdInsP2 by phospholipase C is not appreciably stimulated by ADP. These findings were compared with those obtained when platelets were aggregated by ADP in a medium without added of Ca2+ in which secondary aggregation associated with thromboxane A2 (TXA2) formation and release of granule contents occurs. At 10 s (during primary aggregation) the changes were similar in the two media. At 30 s and 60 s (during secondary aggregation in the low-Ca2+ medium), the increases in PtdInsP2, PtdInsP and phosphatidic acid in platelets suspended in the absence of added Ca2+ were larger than those in platelets suspended in the presence of 2 mM Ca2+. In the absence of added Ca2+, ADP-induced increases in the labelling of InsP3, InsP2 and InsP which were probably due to the effects of TXA2 since they were abolished by aspirin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytotoxicity and genotoxicity testing of sodium fluoride on Chinese hamster V79 cells and human EUE cells.

The cytotoxic effects of sodium fluoride (NaF) on hamster V79 cells and human EUE cells were studied by measuring the cloning efficiency and DNA, RNA and protein synthesis in cells cultured in the presence of NaF. Potential mutagenicity of NaF was followed on the basis of induced 6-thioguanine-resistant mutants in treated Chinese hamster V79 cells. The results showed that the addition of 10-150 micrograms of NaF per ml of culture medium induced 10-75% cytotoxic effect on hamster V79 cells but had no toxic effect on human EUE cells. NaF was cytotoxic to human EUE cells at considerably higher concentrations (200-600 micrograms/ml). Growth of both cell types with 100 and 200 micrograms of NaF per ml caused inhibition of 14C-thymidine, 14C-uridine and 14C-L-leucine incorporation. This means that NaF inhibits macromolecular synthesis whereby damaging effects were less drastic in human EUE cells. The results of detailed mutagenicity testing on hamster V79 cells showed that NaF did not show any mutagenic effect after long-term (24-h) incubation of hamster cells in the presence of 10-400 micrograms of NaF per ml of culture medium.     

Characterization and immunocytochemical localization of lipophorin binding sites in the oocytes of Rhodnius prolixus

Purified lipophorin, metabolically labelled with 32P exclusively in the phospholipid moiety, was used to study the process of phospholipid delivery to the oocyte. The kinetics of phospholipid transfer "in vitro," from lipophorin to the oocytes, was linear at least up to 4 h and was impaired by low temperature. A net transfer of phospholipids from lipophorin particles to the oocytes was observed. The rate of phospholipid uptake was dependent on the concentration of lipophorin in the medium and was shown to be a saturable process. The addition of a molar excess of purified unlabelled lipophorin to the culture medium resulted in a substantial decrease in the transfer of [32P]phospholipids, but no reduction occurred in the presence of a molar excess of albumin. The lipophorin binding sites were localized in the oocytes by immunogold techniques using two different protocols for oocyte fixation. Strong labelling was observed especially at the microvilli. No labelling was detected in the yolk granules.     

Arachidonic Acid Incorporation and Redistribution in Human Neuroblastoma (SK-N-BE) Cell Phospholipids

The incorporation and redistribution of [1-14C]arachidonic acid in SK-N-BE human neuroblastoma cell phospholipids were investigated. By continuous labelling in serum-enriched medium, a rapid radioactivity incorporation into phosphatidylcholine (PtdCho), phosphatidylinositol, and phosphatidylserine was observed; initially, phosphatidylethanolamine (PtdEtn) was poorly labelled, but at later stages it displayed the highest level of arachidonic acid incorporation, in comparison with other phospholipid classes. Labelling of triacylglycerols was also observed. When cells were pulse-labelled with [1-14C]arachidonic acid and then reincubated in label-free medium, a decrease of the radioactivity in triacylglycerols was observed initially, paralleled by an increase of phospholipid labelling; thereafter, arachidonic acid redistribution was consistent with a net decrease of the radioactivity associated with PtdCho acid-stable forms (i.e., diacyl plus alkylacyl forms), concomitantly with a net labelling increase of both acid-stable PtdEtn and alkenylacyl-PtdEtn. Data indicate the following: (a) neuroblastoma cells incorporate arachidonic acid into phospholipids through complex kinetics involving transfer of the fatty acid from acid-stable PtdCho to both alkenylacyl-PtdEtn and acid-stable PtdEtn; and (b) triacylglycerols act as storage molecules for arachidonic acid which is subsequently incorporated into phospholipids. The possibility that arachidonic acid transfer to PtdEtn subclasses is driven by distinct mechanisms is discussed.     

Early inhibition of phospholipid synthesis in dimethyl sulfoxide (DMSO) treated Friend erythroleukemia (FL) cells.

Phosphate metabolism in Friend erythroleukemia cells undergoing DMSO-induced differentiation was studied. Thirty minutes after the cells were exposed to DMSO in medium at pH 7, an inhibition of 39% in the incorporation of phosphate into phospholipids was observed. This decrease was not due to a change in the precursor pools since phosphate uptake and the phosphorylation of the organic soluble compounds were only inhibited 13%. Inhibition of phospholipid synthesis preceded inhibition of RNA and protein synthesis and reached a maximum after 24 hours of DMSO treatment. At this time, the phospholipid content of the cells was also decreased as compared to that of the control untreated cells. Phospholipid synthesis remained at a level significantly lower than in the controls over the 4-day observation period, at which time 85% of the treated cells were benzidine positive. Separation of the different phospholipids by chromatography on thin layer silicate gel plates showed that, after one hour of DMSO treatment, more than 80% of the radioactivity was in phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine. Phosphatidylethanolamine was the most inhibited. Incorporation of inositol into phospholipid was also significantly decreased. However, there was little variation in the phospholipid composition of the treated and non-treated cells other than a decrease in the percent of sphingomyelin after 48 hours of DMSO treatment. These changes in phospholipid metabolism may initiate the first step in the complex differentiation process. The phospholipids are important components of membranes and the inducers are known to influence their fluidity.     

Independent forms of potentially lethal damage fixed in plateau-phase Chinese hamster cells by postirradiation treatment in hypertonic salt solution or araA

Plateau-phase Chinese V79 hamster cells were sequentially treated after exposure to gamma rays in medium made hypertonic by the addition of sodium chloride (370 mM) and with various concentrations of 9-beta-D-arabinofuranosyladenine (araA) to study their combined effect on fixation of potentially lethal damage (PLD). A 10-min treatment in hypertonic medium fixed an extensive amount of PLD and caused a decrease in D0 from 1.8 to 1.2 Gy without significantly affecting Dq. Subsequent treatment with araA caused further fixation of PLD but resulted in a specific, concentration-dependent reduction in Dq from 4.9 to 1.6 Gy after a 4-h exposure to 150 microM araA. A 30-min treatment in hypertonic medium reduced not only Do (from 1.8 to 1.0 Gy) but also Dq (from 4.9 to 2.7 Gy). Subsequent treatment with araA in this case affected only the residual shoulder, reducing it to 1.6 Gy after a 4-h treatment with 100 microM araA, a value similar to that obtained after treatment with araA of cells exposed to salt for only 10 min. When the repair of PLD fixed by a 10-min treatment with salt was measured by delaying its postirradiation application in the presence of various amounts of araA, a small decrease in the repair rate was observed but no significant effect on the relative increase in survival. Qualitatively similar results were obtained for repair of PLD sensitive to araA after a 10-min treatment in hypertonic medium. These results suggest the radiation induction of forms of PLD with different sensitivity to fixation by postirradiation treatments. araA is proposed to fix a form of PLD termed alpha-PLD, the repair of which takes place within 4-6 h and which causes the formation of the shoulder in the survival curve of cells plated immediately after irradiation. Short treatments in hypertonic medium (less than 10 min) are proposed to fix a form of PLD termed beta-PLD, the repair of which takes place within 1 h and leads to restoration of the slope to values equal to those obtained in the survival curve of cells plated immediately after irradiation. However, longer treatments in hypertonic medium also affect Dq and thus also alpha-PLD. Repair of beta-PLD was not significantly affected by araA and repair of alpha-PLD was not significantly affected by short hypertonic treatment, thus indicating the independence of the two forms of PLD.(ABSTRACT TRUNCATED AT 400 WORDS)     

Change of lipid composition of Zygosaccharomyces rouxii after transfer to high sodium chloride culture medium

The change of the lipid composition was investigated in Zygosaccharomyces rouxii cells transferred from NaCl-free to 2 M NaCl medium. After the transfer thesterol and phospholipid contents increased, and phospholipids with higher oleic acid contents and higher negative charge increased. These changes of membrane lipid may be required for the adaptation of Z. rouxii cells to a high NaCl environment.     

Depletion of arachidonic acid from GH3 cells. Effects on inositol phospholipid turnover and cellular activation.

We have adapted rat pituitary GH3 cells to grow in delipidated culture medium. In response, esterfied linoleic acid and arachidonic acid become essentially undetectable, whereas eicosa-5,8,11-trienoic acid accumulates and oleic acid increases markedly. These changes occur in all phospholipid classes, but are particularly pronounced in inositol phospholipids, where the usual stearate/arachidonate profile is replaced with oleate/eicosatrienoate (n - 9) and stearate/eicosatrienoate (n - 9). Incubation of arachidonate-depleted cells with 10 microM-arachidonic acid for only 24 h results in extensive remodelling of phospholipid fatty acids, such that close-to-normal compositions and arachidonic acid content are achieved for the inositol phospholipids. In comparison studies with arachidonic acid-depleted or -repleted cells, it was found that the arachidonate content does not affect thyrotropin-releasing-hormone (TRH)-stimulated responses measured at long time points, including [32P]Pi labelling of phosphatidylinositol and phosphatidic acid, stimulation of protein phosphorylation, and basal or TRH-stimulated prolactin release. However, transient events such as stimulated breakdown of inositol phospholipids and an initial rise in diacylglycerol are enhanced by the presence of arachidonate. These results show that arachidonic acid itself is not required for operation of the phosphatidylinositol cycle and is not an obligatory intermediate in TRH-mediated GH3 cell activation. It is possible that any structural or functional role of arachidonic acid in these processes is largely met by replacement with eicosatrienoate (n - 9). However, since arachidonate in inositol phospholipids facilitates their hydrolysis upon stimulation by TRH, arachidonic acid apparently may have a specific role in the recognition of these lipids by phospholipase C.     

Changes in the lipid composition of murine neuroblastoma C 1300 cells during the process of differentiation

The paper deals with the lipid composition of nondifferentiated cells at the logarithmic and stationary growth phases as well as with differentiated cells of C 1300 neuroblastoma adopted to the Eagle medium. It is shown that phospholipids and cholesterol dominate at the studied growth phases among lipids. Their amount in the differentiation process is thrice as high calculating per cells. The quantity of individual phospholipids changes in different manner during differentiation. The phosphatidylinosite level is higher than of other substances.