Inhibition of plasma membrane NADH oxidase activity and growth of HeLa cells by natural and synthetic retinoids

Several retinoids, both natural and synthetic, were evaluated for their ability to modulate NADH oxidase activity of plasma membranes of cultured HeLa cells and the growth of HeLa cells in culture. Both NADH oxidase activity and the growth of cells were inhibited by the naturally-occurring retinoids all trans-retinoic acid (tretinoin) and retinol as well as by the synthetic retinoids, trans-acitretin, 13-cis-acitretin, etretinate and arotonoid ethylester (Ro 13-6298). For all retinoids tested, inhibition of NADH oxidase activity and inhibition of growth were correlated closely. With tretinoin, etretinate and arotonoid ethylester, NADH oxidase activity and cell growth were inhibited in parallel in proportion to the logarithm of retinoid concentration over the range of concentrations 10-8 to 10-5 M. Approximately 70% inhibition of both NADH oxidase activity and growth was reached at 10 µM. With retinol, trans-acitretin and 13-cis-acitretin, inhibition of NADH oxidase activity and growth also were correlated but maximum inhibition of both was about 40% at 10 µM. The possibility is suggested that inhibition of the plasma membrane NADH oxidase activity by retinoids may be related to their mechanism of inhibition of growth of HeLa cells in culture. (Mol Cell Biochem 166: 101-109, 1997)     

Differential Sensitivity of Telomerase from Human Hematopoietic Stem Cells and Leukemic Cell Lines to Mild Hyperthermia

We have investigated the effects of hyperthermia (HT) on cell proliferation and telomerase activity of human hematopoietic stem cells (HSCs) and compared with human leukemic cell lines (TF-1, K562 and HL-60). The cells were exposed to HT at 42 and 43 °C up to 120 min. The cells were incubated at 37 °C for 96 h. Then the cells were collected and assayed for cell proliferation, viability, telomerase activity, and terminal restriction fragment (TRF) lengths. The enzyme activity from HSCs was decreased up to 68.6 at 42 and 85.1 % at 43 °C for 120 min. This inhibition in leukemic cells was up to 28.9 and 53.6 % in TF-1; 53 and 63.9 % in K562; 45.2 and 61.1 % in HL-60 cells. The treated cells showed TRF lengths about 5.3 kb for control HL-60 cells, 5.0 kb for HL-60 cells treated at 42 and 4.5 kb at 43 °C for 120 min. In HSCs, the TRF length was about 4.5 kb for untreated cells and 4.0–4.5 kb for treated cells at 42 and 43 °C for 120 min. The time response curves indicated that, inhibition of the enzyme activity in leukemic cells was dependent to the time of exposure to HT. But in HSCs, the inhibition was reached to steady state at 15 min exposure to 43 °C heat stress. TRF length was constant at treated two types of cells, which implies that in cells subjected to mild HT no telomere shortening was observed.     

Retinoid suppression of transglutaminase activity and envelope competence in cultured human epidermal carcinoma cells

Growth of SCC-13 squamous carcinoma cultures in the presence of retinoids considerably reduced the expression of two differentiation markers, the cellular capability to form cross-linked envelopes, and the enzyme transglutaminase required for cross-linking. A limited survey of retinoids showed that all-trans retinoic acid, 13-cis retinoic acid, and arotinoid Ro 13-6298 were highly effective in the absence of hydrocortisone and were only slightly antagonized by its presence in the medium. In contrast, retinyl acetate, retinol, and retinol bound to its plasma binding protein were quite active in the absence of hydrocortisone but were essentially inactive in its presence. Dexamethasone was also highly effective in antagonizing the suppressive action of retinyl acetate on envelope formation, while the corticosteroid antagonists cortexolone and progesterone were inactive. These results suggest that there are separate pathways, which are differentially regulated by hydrocortisone, for either the metabolism or action of retinol and retinoic acid in SCC-13 cells.     

Anticancer effects of 6-<Emphasis Type="Italic">o</Emphasis>-palmitoyl-ascorbate combined with a capacitive-resistive electric transfer hyperthermic apparatus as compared with ascorbate in relation to ascorbyl radical generation

The aim of the present study is to determine the anti-proliferative activity of 6-o-palmitoyl-l-ascorbic acid (Asc6Palm) that is a lipophilic derivative of l-ascorbic acid (Asc), on human tongue squamous carcinoma HSC-4 cells by combined use of hyperthermia in comparison to Asc. Asc6Palm or Asc were administered to HSC-4 cells for 1 h, to which hyperthermia at 42 °C was applied for initial 15 min. After further 1–72 h incubation at 37 °C, cell proliferation was determined with Crystal Violet staining. Ascorbyl radical (AscR) in HSC-4 cell suspension was measured by electron spin resonance (ESR), and cell morphology was observed with scanning electron microscopy (SEM). At 37 °C, 4 mM Asc or 0.35 mM Asc6Palm were enough to suppress proliferation of HSC-4 cells. By combined use of hyperthermia at 42 °C, cell proliferation was decreased when compared to 37 °C. After Asc of 4 mM was incubated with HSC-4 cell suspensions at 37 °C or 42 °C for 0–180 min, the signal intensity of ascorbyl radical (AscR) by ESR was not different regardless of the presence or absence of cells at 37 °C, whereas AscR signal was enlarged in the presence of HSC-4 cells at 42 °C. It was suggested that oxidation of Asc occurred rapidly in HSC-4 cells by hyperthermia, and thereby enhanced the anti-proliferative activity. By SEM observation, the surface of HSC-4 cells treated with Asc6Palm revealed distinct morphological changes. Thus, the combined regimen of Asc6Palm and hyperthermia is expected to exert a marked antitumor activity.     

Effect of temperature on extracellular accumulation of beta-D-N-acetylglucosaminidase in I-cell disease fibroblast cultures.

The activities of most lysosomal enzymes are elevated in the culture fluid of skin fibroblasts derived from patients with I-cell disease with a corresponding reduction in the intracellular activities when the cells are cultured at 37°C. When I-cell fibroblast cultures are incubated at 27°C for 8–24 hr, the β-D-N-acetylglucosaminidase (EC 3.2.1.30) activity accumulated in the culture fluid is reduced to approximately 25% of the activity in 37°C control cultures without a corresponding change in intracellular activity. No significant effect of temperature is observed on the intra- and extracellular distribution of β-D-N-acetylglucosaminidase in non-I-cell fibroblast cultures. These findings suggest the existence of two lysosomal enzyme pools in I-cell fibroblasts, one of which is temperature-dependent and destined for excretion while the other remains intracellular and appears to be unaffected by temperature.     

Sedimentation equilibrium in reacting systems. VII. The temperature-dependent self-association of beta-lactoglobulin A at pH 2.46

The polyene antibiotic filipin inhibits the activities of both photosystem I and photosystem II in maize mesophyll chloroplasts and pea chloroplasts. Maximum inhibition of photosystem II activity was observed at a filipin concentration of about 0.4 mm in maize mesophyll chloroplasts and 1.0 mm in pea chloroplasts. Inhibition of photosystem II activity was temperature dependent, being much less if the antibiotic and chloroplasts were incubated at 0 °C compared to 25 °C. The inhibition of photosystem I activity of both maize mesophyll and pea chloroplasts caused by filipin, could be overcome by the addition of the soluble electron transfer protein, plastocyanin. It is concluded that the inhibition of photochemical activity caused by filipin is a secondary effect resulting from a change in membrane conformation induced by the antibiotic.     

Increase in Shedding of Intercellular Adhesion Molecule-1 in Human Malignant Melanoma Cell Lines Treated With Hyperthermia In Vitro

Human malignant melanoma cell lines were found to increase shedding of soluble intercellular adhesion molecule-1 (sICAM-1) into the culture medium when the cells were treated with hyperthermia at 41–43°C for 3–6 hr in vitro. The content of ICAM-1 in the cell lysate was also found to be increased after hyperthermia. The increased rate of ICAM-1 concentration in the cells was at maximum when they were incubated at 41°C for 3 hr. Also, the melanoma cell lines heat-treated at 41°C showed more intense immuno-fluorescence in the ICAM-1 expression on the cell surface. It remains to be investigated further whether the effects of hyperthermia on the ICAM-1 expression in melanoma cells is to augment membrane ICAM-1 expression, which in turn leads to shedding of soluble ICAM-1 or only to acceleration of shedding of sICAM-1 by unknown mechanisms.     

The release of cytosolic proteins from cells treated with concanavalin A during scraping from plastic culture dishes

When rat hepatoma cells (HTC and R117-21B), treated with concanavalin A (conA) at 37 °C, were scraped from plastic culture dishes with a silicone-rubber policeman, the cell membranes were broken and the cytoplasm was released. This phenomenon was also observed in cells treated with conA at 4 °C, even though it took a longer time to show the same effect. The effect of 10 μg/ml of conA on the release of the cellular proteins reached a plateau when the treatment was carried out at 37 °C. Ninety percent of this effect was abolished by 10 mM of α-methyl-d-mannoside. The effect was completely nullified by 100 mM. At 4 °C, however, even 100 mM of this sugar could not abolish this effect. The apparent decrease in the cellular proteins with conA after scraping was observed not only in the logarithmic phase, but also in the stationary phase of cell growth. The breakdown of plasma membranes with conA eventually caused decrease in tyrosine aminotransferase activity, even though the lectin induced the enzyme activity in cultured cells.     

Induced pore formation in membranes of cortex cells in excised Sorghum roots: its effect on membrane potential and nucleotide leakage

Measurements of membrane potentials were used for screening various molecules capable of inducing pore formation in cellular membranes for their ability to penetrate into excised roots of Sorghum bicolor (L.) Moench.
Poly- l -lysine (30 000 MW) and amphotericin B did not affect cortical cells even though they affected epidermal cells; the lysine polymer and the amphotericin B micelle were apparently too large to diffuse into the roots. Polymyxins and filipin depolarized the epidermal cells as well as the cells of the cortex. Depolarization of cortical cells by filipin was slower than that by polymixins.
Leakage of adenylate nucleotides was measured to obtain information concerning the size of the pores induced by polymyxins. Incubation with 200 μg/ml polymyxin B caused leakage of ATP from root segments. Leaked nucleotides were found in the medium; however, with time they were dephosphorylated, presumably by cell wall phosphatases. In situ ATP formation was observed when root segments were incubated with polymyxin, ADP, Pi, succinate and glucose, confirming that the induced pores were larger than the size of a nucleotide.     

Effects of lipid-phase separation on the filipin action on membranes of ergosterol-replaced Tetrahymena cells,as studied by freeze-fracture electron microscopy

The effects of lipid-phase separation on the filipin action on pellicle membranes of ergosterol-replaced Tetrahymena pyriformis cells were studied by freeze-fracture electron microscopy. The pellicle membranes with phase separations induced by chilling from 34°C (growth temperature) to lower temperatures (30, 22 and 15°C) were treated with filipin. This produced filipin-induced lesions (“pits”) only in the particulated (liquid) regions along the margin between solid and liquid domains, while they were produced in the particle-free (solid) areas when membranes were chilled to 15°C. The pellicle membranes with lesions induced by filipin at 34°C were chilled to 22°C. This chilling raised larger particle-free areas and more condensed particle-aggregations on the membranes than on the membranes without the filipin treatment. These results suggest that the membrane fluidity affects induction and development of the ergosterol-filipin complex in the membrane.     

Generation of nitric oxide by human neutrophils

Human neutrophils were evaluated for their ability to generate nitric oxide. Neutrophils incubated with superoxide dismutase at 37 degrees C produce nitrite anion at a rate of 1.8 nmols/2 x 10(6) cells/30 min, providing indirect evidence of nitric oxide production. Incubation of the neutrophils with concentrations of serum-opsonized zymosan, N-formyl-methionyl-leucyl-phenylalanine, or phorbol myristate acetate sufficient to stimulate the respiratory burst and lysosomal enzyme release caused no additional nitrite anion production. Glass wool-adherent neutrophils exhibited a similar dissociation of nitrite anion production from the respiratory burst and lysosomal enzyme release. Direct evidence for nitric oxide production was also obtained using nitric oxide-specific chemiluminescence. These results demonstrate that human neutrophils are capable of generating nitric oxide.     

Effects of cytochalasin D on fusion of cells by HVJ (Sendai virus).

Fusion of cells mediated by HVJ was inhibited completely with 5 μg/ml or more of cytochalasin D (CD). With cytochalasin, HVJ-cell interaction at 0 °C proceeded as well as without cytochalasin; HVJ was adsorbed to cell surfaces and the cells agglutinated together. Then the virus particles were enfolded with cell membranes, which resulted in the disappearance of hemadsorption activity on the cell surfaces. When the cell-virus complex was incubated at 37 °C, the early reactions proceeded as well as without cytochalasin; the hemadsorption activity reappeared on the cell surfaces, the viral envelopes fused with cell membranes at the same degree as without cytochalasin, and a stage sensitive to sodium azide appeared as in a control without cytochalasin. But cell-to-cell fusion did not occur in the presence of cytochalasin; cells were dissociated freely from the cell aggregates during incubation. This indicates that cell-to-cell fusion was inhibited but HVJ envelope to cell membrane interactions proceeded well on incubation at 37 °C. These findings suggest that viral envelope-cell membrane fusion and cell-cell fusion are separable, and participation of a cytoskeleton system including microfilaments in the cells is essential for cell-cell fusion.     

Concanavalin a promotes the uptake of lysosomal hydrolases by human fibroblasts

Human placental hexosaminidase B and β-galactosidase are taken up very poorly by human fibroblasts in culture. However, if fibroblasts manifesting genetically determined deficiencies of these lysosomal hydrolases are first treated with concanavalin A, then enzyme uptake is markedly increased. Enzyme activity which becomes associated with concanavalin A-treated fibroblasts maintained at 4°C can be greatly removed by treatment with haptene sugar, while enzyme activity which becomes associated with cells maintained at 37°C is refractory to haptene treatment. These results are interpreted as an initial binding of enzyme to concanavalin A molecules located at the cell surface, followed by an active cellular process leading to internalization of the lectin-enzyme complexes.     

Activation of a ribonuclease on dispersion of rat liver to a single cell suspension and incubation of the cells at 37 degrees

Incubation of rat liver parenchymal cell suspensions at 37° results in degradation, to acid-soluble material, of 15% of cellular RNA at 30 minutes and 40% at three hours, beyond which there is little, if any, further degradation. The RNA which remains in the acid-insoluble form in the cells up to 30 minutes appears to exist largely in the native state. However, after 30 minutes, the acidinsoluble RNA of the cells is found to be partially depolym-erised. These observations suggest the activation of an intracellular nuclease on dispersion of the liver tissue to a single cell suspension and incubation at 37°. This nuclease appears to be responsible also for the degradation, reported earlier, of exogenous RNA taken up by the cells. Activation of the nuclease is not due to depletion of pool of ATP or of other ribonucleotides from the cells, during either dispersion of the tissue or incubation at 37°. Incubation of the cells at 28°, or of liver slices at 37°, does not lead to any significant degradation to acid-soluble material, or to partial depolymerisation, of RNA. Analysis of RNA obtained from cells incubated at 37° for various periods showed that chromatography on methylated albumin-kiesulguhr (MAK) and Sephadex columns is not suitable for detecting partial depolymerisation of cellular RNA; RNA shown to be partially depolymerised by analysis on sucrose density gradient, in an analytical ultracentrifuge, and on a cellulose column, gave the normal pattern in MAK or Sephadex runs.     

High retinoid X receptor expression in JEG-3 choriocarcinoma cells: Involvement in cell function modulation by retinoids

Retinoic acid (RA) is an important mediator of cell differentiation. It stimulates hCG secretion by JEG-3 choriocarcinoma cells in vitro after a time lag. The first aim of this study was to characterize which types of retinoid receptors (RARs and RXRs) are present in JEG-3 cells. Using Western blot analysis and immunocytochemistry with specific antibodies as well as Northern blot analysis, we found that JEG-3 cells expressed RARα and RXRα, the latter being the predominant receptor. We then analyzed the action on cell proliferation and hCG secretion of the physiological retinoids all-trans RA (RA) and 9 cis RA as well as synthetic retinoids with specific affinity for RARα and RXRα. All these retinoids were potent inhibitors of cell growth, maximal inhibition (72 ± 2%) being observed after 4 days of treatment with Ro 25, a RXRα specific ligand. Within 24 h, 9 cis RA and Ro 25 stimulated hCG secretion, and maximal stimulation (1,472 ± 10%) occurred at 48 h with the RXRα-specific ligand. The RARα-specific ligand also stimulated hCG secretion but to a lower extend and after a delay of 48 h. These results suggest a predominant role of RXRα in mediating the biological effects of retinoids on JEG-3 cells and the possible induction by RA itself of the metabolic pathway leading to 9 cis RA. J. Cell. Physiol. 176:595–601, 1998. © 1998 Wiley-Liss, Inc.     

The role of energy in hyperthermia-induced mammalian cell inactivation: A study of the effects of glucose starvation and an uncoupler of oxidative phosphorylation

When cultured Chinese hamster cells were exposed to 43°C hyperthermia, effects due to glucose deprivation and to the presence of the uncoupler of oxidative phosphorylation, carbonylcyanide-3-chlorophenylhydrazone, during the 43°C treatment proved to be strongly accelerated compared to the effects at normal temperature (37°C). This strongly indicates that the availability of energy plays an important role in the response of these cells to hyperthermia. One of the reasons cells die after hyperthermia may be a lethal lack of energy. Cells heated before glucose deprivation were able to maintain viability for a longer period during deprivation than cells without the preheat treatment. As the cells might develop thermotolerance after the heat exposure, this suggests that cells in the thermotolerant state use energy in a more economical way.     

Retinoid-induced inhibition of eosinophil LTC4 production

Naturally occurring and synthetic retinoids demonstrate a marked antiinflammatory effect when employed in such disorders as acne and psoriasis. This effect may result in part from their inhibition of release of potent mediators (e.g. eicosanoids) by inflammatory cells. In this study, we examined the effect of eight retinoids (tretinoin, isotretinoin, retinol, retinal, acitretin, retinyl palmitate, etretinate, Ro 15-0778) on the release of leukotriene (LT)C4, an important lipid mediator generated by eosinophils. Tretinoin, isotretinoin, retinol, retinal, and acitretin at 10(-5) M or 10(-4) M concentrations inhibited LTC4 release by A23187-stimulated horse eosinophils in vitro; 10(-4) M retinyl palmitate was also inhibitory. However, 10(-5) M etretinate augmented A23187-induced LTC4 release, and the arotinoid Ro 15-0778 had no effect on LTC4 production. These data suggest that selected retinoids may have potential use in the reduction of LTC4 generation by eosinophils. This inhibition could be beneficial in the therapy of such diseases as bronchial asthma in which release of LTC4 may be involved in the inflammatory process.     

Cellular ion content changes during and after hyperthermia.

The potassium (K) level in mouse mastocytoma P815 cells undergoes a 40% reduction within 30 minutes of incubation at 43°C. It decreases further when the cells return to 37°C after a 60 minute 43°C incubation. A smaller change (20%) occurs after a 60 minute incubation at 41°C. Furthermore, nearly all of the lost K recovers in two hours after a subsequent incubation at 37°C. On the other hand, the sodium level in the cells increases by an amount much smaller than the potassium changes. However, the net loss of cations from the cells undergoing hyperthermia does not induce a simultaneous reduction of intracellular water volume.     

Heat shock induces simultaneous rearrangements of all known cytoskeletal filaments in normal interphase fibroblasts

Hyperthermia (heat shock (HS)) induces changes in morphology of nucleoli, cytoplasmic organelles, and cytoskeleton. Responses to hyperthermia are, as a rule, similar in all types of eukaryote cells. However, there is no information on the uniformity of the cytoskeleton heat shock response (CHSR) in different cell types. This has led to the conclusion that the eukaryote CHSR depends on the cell type. We studied CHSR only in one cell type-in normal embryonic mouse fibroblasts (NEMFs) and in normal embryonic rat fibroblasts (NERFs), as well as in normal postnatal rat fibroblasts (NPRFs), by using the method of fluorescence microscopy. Incubation of the cells at 43°C led to a rearrangement of cytoskeleton. Responses of cytoskeleton to HS in NEMF, NERF, and NPRF were similar. Heat shock resulted in disassembly of bundles of actin filaments (AFs), marked changes in microtubule (MT) morphology, and collapse of intermediate filaments (IFs) around the nucleus. Rearrangements of different cytoskeleton filament types occurred simultaneously and were seen as soon as after 2–4 min. After 30–120 min of incubation at 43°C, the cells were still capable of rebuilding the actin cytoskeleton after the temperature had returned to normal (37°C). We believe that the cytoskeleton rearrangement under the action of HS can be of vital importance in cell protection against temperature stress. Data are discussed on possible coupling of the CHSR process with rearrangement of the protein synthesizing system, which leads to initiation and/or stimulation of synthesis of HS proteins.