Cytotoxicity of lazaroid U-75412E in human epithelial cell line (Wish).

The 21-aminosteroids (or lazaroids) are a recently synthesized class of compounds demonstrated to protect tissue against damage induced by trauma and/or ischemia. Currently, very little is known about the biological effects of lazaroids. In this work the action of lazaroid U-75412E on a human epithelial cell line (Wish) was evaluated. The data obtained showed an inhibition of cell growth and a dose- and time-dependent decrease of cell viability. Furthermore, a dose- and time-dependent increase of cells in the G2/M phase with the appearance of apoptotic cells was observed by flow cytometric analysis. Nuclear fragmentation was also evident. Lactate dehydrogenase release and scanning electron microscopy experiments suggested that plasma membrane integrity was altered by this compound. The immunofluorescence technique and transmission electron microscopy images also showed intracellular damage, such as alteration of microtubular arrangement, mitochondrial swelling and the presence of vacuoles. This study demonstrated that 1 microM U-75412E was unable to modify these parameters, while higher concentrations (6-75 microM) had a cytotoxic effect on Wish cells.

Dexamethasone apoptosis induction and glucocorticoid receptor levels in cultured normal and neoplastic cell lines.

The deletion with apoptotic mechanisms, of different normal and neoplastic cell lines [Jurkat leukemic cells, EUE epithelioid cells, normal (FG) and transformed rat fibroblasts (SGS/3A)] cultured in vitro in presence of dexamethasone, have been studied combining morphocytochemical (fluorescence microscopy), cytometric (flow cytometry) and biochemical (Radio Receptor Assay) analyses. It has been found that the synthetic glucocorticoid hormone induces an antiproliferative effect with accumulation of cells in the G1 phase and a cell loss by programmed death in some cell lines. The apoptotic incidence was found to be inversely proportional to the cytostatic effect of the hormone: the highest in EUE and Jurkat cells (in EUE cells not only affecting the G0/G1 phase of the cell cycle), the lowest in SGS/3A cells and absent in frbroblasts FG. The apoptotic degeneration, in all the cell lines studied, was characterized, morphologically and cytochemically by: a) decrease in stainability/content of cell DNA and proteins; b) condensation of cytoplasm; c) preservation of mitochondrial membrane functional integrity. In conclusion, in the presence of dexamethasone, programmed cell death was found to play a variable role during the maintenance of culture turnover in different cell lines and the incidence of degenerative phenomena does not appear to be related to glucocorticoid receptor levels.

Role of oxidative stress in the manganese and 1-methyl-4-(2'-ethylphenyl)-1,2,3,6-tetrahydropyridine-induced apoptosis in PC12 cells.

Oxidative stress is thought to play a key role in the apoptotic death of several cellular systems, including neurons. Oxidative stress is proposed also as a mechanism of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- and manganese (Mn)-induced neuronal death. We have recently shown that Mn and the MPTP analogue 1-methyl-4-(2'-ethylphenyl)-1,2,3,6-tetrahydropyridine (2'Et-MPTP), which is metabolized by MAO-A to 1-methyl-4-(2'-ethylphenyl)-pyridinium ion, induce apoptosis in PC12 cells. In the present study, we evaluated the effects of deprenyl and the antioxidant drugs N-acetylcysteine (NAC) and ascorbic acid (AA) on Mn- and 2'Et-MPTP-induced apoptosis in PC12 cells. Apoptosis was tested by terminal deoxynucleotidyl transferase-mediated 2'-deoxy-uridine-5'-triphosphate nick end labelling (TUNEL) technique, flow cytometry and fluorescence microscopy. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Mn-induced apoptosis and decrease in cell viability was inhibited by the antioxidants NAC and AA. Deprenyl failed to inhibit the above Mn effects. Neither NAC, AA nor deprenyl were able to inhibit both 2'Et-MPTP-induced apoptosis and decrease in cell viability. These results confirm that apoptosis may be an important mechanism of cell death in MPTP- and Mn-induced parkinsonism. However, an oxidative stress mechanism may be recognized, at least in vitro, only in the Mn-induced apoptosis.

Manganese and 1-methyl-4-(2'-ethylpheny1)-1,2,3,6-tetrahydropyridine induce apoptosis in PC12 cells.

Oxidative stress is thought to play a key role both in the neurotoxin MPTP- and manganese (Mn)-induced neurotoxicity and in apoptotic cell death. In the present study, we report that Mn and the MPTP analogue 1-methyl-4-(2'-ethylphenyl)-1,2,3,6-tetrahydropyridine (2'Et-MPTP), which is metabolized by MAO-A to 1-methyl-4-(2'-ethylphenyl)-pyridinium ion (at concentrations of 0.5 and 1.0 mM), induced apoptosis in PC12 cells. Apoptosis was tested by terminal deoxynucleotidyl transferase-mediated 2'-deoxy-uridine-5'-triphosphate nick end labelling (TUNEL) technique, flow cytometry and fluorescence microscopy. Both Mn and 2'Et-MPTP induced also a time-dependent decrease in cell viability, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Only Mn-induced apoptosis and decrease in cell viability were inhibited by the antioxidant ascorbic acid. We conclude that apoptosis may be an important mechanism of cell death in MPTP- and Mn-induced parkinsonism. However, an oxidative stress mechanism may be recognized only in the Mn-induced apoptosis.

Protective effect of deferoxamine on sodium nitroprusside-induced apoptosis in PC12 cells.

Reportedly, the generation of nitric oxide (NO) may lead to iron mobilization from ferritin disrupting intracellular iron homeostasis and increasing levels of reactive oxygen species. In the present study, we evaluated the role of endogenous iron in NO-induced apoptosis in PC12 cells. Apoptosis was tested by flow cytometry, fluorescence microscopy and terminal deoxynucleotidyl transferase-mediated 2'-deoxy-uridine 5'-triphosphate nick end labeling (TUNEL) technique. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. When incubated with 0.5-0.75 mM sodium nitroprusside (SNP, a chemical NO donor), PC12 cells were shown to undergo apoptosis. In addition, SNP induced a time-dependent decrease in cell viability. Since deferoxamine (0.05-0.1 mM), a powerful iron chelator, inhibited both SNP-induced apoptosis and the decrease in cell viability, we suggest that these NO effects may be dependent upon iron mobilization within the cell.

Activation signals of T lymphocytes in microgravity.

Human peripheral blood lymphocytes and monocytes were activated with concanavalin A with or without exogenous recombinant interleukin 1 (IL-1) alone or IL-1 + interleukin 2 (IL-2) under microgravity conditions to test the hypothesis that lack of production of IL-1 by monocytes is the cause of the near total loss of activation observed earlier on several Spacelab flights. The 60 min failure of the on-board 1 x g reference centrifuge at the time of the addition of the activator renders the in-flight data at 1 x g unreliable. However, the data from a previous experiment on SLS-1 show that there is no difference between the results from the in-flight 1 x g centrifuge and 1 x g on ground. The comparison between the data of the cultures at 0 x g in space and of the synchronous control at 1 x g on ground show that exogenous IL-1 and IL-2 do not prevent the loss of activity (measured as the mitotic index) at 0 x g; production of interferon-gamma, however, is partially restored. In contrast to a previous experiment in space, the production of IL-1 is not inhibited.

Movements and interactions of leukocytes in microgravity.

The mitogenic activation of human lymphocytes resuspended in vitro is dramatically reduced in microgravity. As cell-cell contacts are one of the elements essential for activation, the behaviour of human leukocytes (mainly lymphocytes and monocytes as accessory cells) in the presence of the mitogen concanavalin A was studied in the centrifuge microscope NIZEMI at 0 x g. Aggregates (formed by intercellular bindings of membrane glycoproteins via the tetravalent alpha-glucoside ligand concanavalin A) were found at 0 x g as well as at 1 x g already 12 h after the addition of the mitogen. In general, the aggregates observed at 0 x g after an incubation time of 46 and 78 h were smaller than the corresponding aggregates in the ground control. The findings are of primary importance since they confirm the indirect evidence we had from earlier Spacelab experiments and demonstrate that cell-cell contacts are occurring also in microgravity. In addition, single cells in 0 x g show a significant higher locomotion velocity than the cells at 1 x g. The fact that the locomotion capability is not decreased during the 78-h incubation with concanavalin A provides further evidence that the cells are not proceeding through the cell cycle.

Dependence of fibroblast autofluorescence properties on normal and transformed conditions. Role of the metabolic activity.

The dependence of autofluorescence properties on the metabolic and functional engagement and on the transformation condition was studied on single cells. Normal Galliera rat fibroblasts at low subculture passage (cell strain), at high subculture passage (stabilized cell line), and transformed cell line derived from a rat sarcoma were used as a cell model. The study was performed by microspectrofluorometric and fluorescence imaging technique. The autofluorescence properties of cells were studied by excitation at two wavelengths, namely 366 nm and 436 nm, that are known to favor the emission of different fluorophores. Spectral shape analysis indicated that under excitation at 366 nm autofluorescence is ascribable mainly to coenzyme molecules, particularly to reduced pyridine nucleotides, while under excitation at 436 nm, flavin and lipopigment emission is favored. The energetic metabolic engagement of the different cell lines was analyzed in terms both of parameters related to anaerobic-aerobic pathways (biochemical assay) and of mitochondrial features (supravital cytometry). The results showed that the cell strain and the stabilized and transformed cell lines can be distinguished from one another on the basis of both overall fluorescence intensity and the relative contributions of spectral components. These findings indicated a relationship between autofluorescence properties and energetic metabolism engagement of the cells that, in turn, is dependent on the proliferative activity and the transformed condition of the cells. In that it is a direct expression of the energetic metabolic engagement, autofluorescence can be assumed as an intrinsic parameter of the cell biological condition, suitable for diagnostic purposes.

Channelling of deoxyribose moiety of exogenous DNA into carbohydrate metabolism: role of deoxyriboaldolase.

In bacteria, the addition of (deoxy)nucleosides or (deoxy)ribose to the growth medium causes induction of enzymes involved in their catabolism, leading to the utilisation of the pentose moiety as carbon and energy source. In this respect, deoxyriboaldolase appears the key enzyme, allowing the utilisation of deoxyribose 5-P through glycolysis. We observed that not only deoxynucleosides, but also DNA added to the growth medium of Bacillus cereus induced deoxyriboaldolase; furthermore, the switch of the culture from aerobic to anaerobic conditions caused a further increase in enzyme activity, leading to a more efficient channelling of deoxyribose 5-P into glycolysis, probably as a response to the low energy yield of the sugar fermentation. In eukaryotes, the catabolism of (deoxy)nucleosides is well known. However, the research in this field has been mainly devoted to the salvage of the bases formed by the action of nucleoside phosphorylases, whereas the metabolic fate of the sugar moiety has been largely neglected. Our results indicate that the deoxyriboaldolase activity is present in the liver of several vertebrates and in a number of cell lines. We discuss our observations looking at the nucleic acids not only as informational molecules, but also as a not negligible source of readily usable phosphorylated sugar.

Relationship between actin microfilaments and plasma membrane changes during apoptosis of neoplastic cell lines in different culture conditions.

In this study we investigated the relationship between the reorganisation of actin cytoskeleton and the changes at cell surface level (i.e. PS exposure and blebbing) in two neoplastic cell lines during apoptosis: Chang liver cells (adherent culture) and promyelocytic HL-60 cells (suspension culture), treated with the podophyllotoxin derivative VP16. The morphological analysis, performed by means of conventional fluorescence microscopy and confocal laser scanning microscopy, on Chang cells showed that onset and progress of the two processes are synchronised. The initial disassembly of stress fibers was associated with the early PS exposure on the cell surface. Moreover, the accumulation of actin at cortical level appeared strongly associated with an intense labelling for Annexin V and, in some cases, especially in the areas of membrane blebbing. The double staining for actin and PS exposure, quantitatively analysed by flow cytometry in HL-60 cells after different treatment times, demonstrated that the decrease of Annexin V binding in the late stages of apoptosis is associated with the strong reduction of actin labelling probably also due to a proteolytic cleavage. These events were also partially related to variations of the functional state of mitochondria, by analysing cytofluorometrically the dissipation of the inner membrane potential (delta psi m).

Cytoskeleton actin changes in IL-2 activated cells.

In the present study we analysed the changes in cytoskeleton actin in lymphoid cells following IL-2 activation and during cell interactions by means of light and electron microscopy, immunofluorescence and molecular analysis. By morphological analysis we observed a higher fluorescence in the activated cells than in the quiescent ones with no modifications in the cytoskeleton pattern comparing activated to resting cells. The results of molecular analysis indicate that, after IL-2 activation, there is a reorganisation of the actin component of the cell cytoskeleton accompanied by the differential expression of the corresponding genes. A future study will be extended to the analysis of others components of the cytoskeleton network.

Static cytofluorometry and fluorescence morphology of mitochondria and DNA in proliferating fibroblasts.

The shape, distribution, and content of mitochondria in individual cells were examined during the cell cycle phases (G(0)/G(1), S, G(2) mitosis) in living human fibroblasts by static cytofluorometry and fluorescence microscopy. The morphocytochemical evaluations were performed in cell cultures submitted to double supravital fluorochrome staining with Hoechst 33342 and DiOC(6) to label DNA and mitochondria, respectively. The staining modalities were based on the stability of mitochondrial labeling. The G(1) to early S phases were characterized by the presence of filamentous mitochondria, except during the early postmitotic period. During late S, G(2), and mitotic phases, mitochondrial mass reached its highest value and mitochondria became short and numerous. During the last stage of mitosis, mitochondria were distributed among daughter cells through a cytoplasmic bridge.

Influence of microgravity on mitogen binding and cytoskeleton in Jurkat cells.

The effects of microgravity on Jurkat cells--a T-lymphoid cell line--was studied on a sounding rocket flight. An automated pre-programmed instrument permitted the injection of fluorescent labelled concanavalin A (Con A), culture medium and/or fixative at given times. An in-flight 1 g centrifuge allowed the comparison of the data obtained in microgravity with a 1 g control having the same history related to launch and re-entry. After flight, the cells fixed either at the onset of microgravity or after a or 12 minute incubation time with fluorescent concanavalin A were labelled for vimentin and actin and analysed by fluorescence microscopy. Binding of Con A to Jurkat cells is not influenced by microgravity, whereas patching of the Con A receptors is significantly lower. A significant higher number of cells show changes in the structure of vimentin in microgravity. Most evident is the appearance of large bundles, significantly increased in the microgravity samples. No changes are found in the structure of actin and in the colocalisation of actin on the inner side of the cell membrane with the Con A receptors after binding of the mitogen.

PMA withdrawal in PMA-treated monocytic THP-1 cells and subsequent retinoic acid stimulation, modulate induction of apoptosis and appearance of dendritic cells.

OBJECTIVES: To analyse proliferation, differentiation and apoptosis in THP-1 cells after stimulation with phorbol 12-myristate 13-acetate (PMA) and retinoic acid (RA). MATERIALS AND METHODS: PMA and RA were used in a three-step-procedure: (i) treatment with 6, 30, 60 nm PMA, that induced initial, intermediate and advanced levels of monocyte-macrophage transition, respectively; (ii) recovery in PMA-free medium; (iii) incubation with 4 µm RA. Cultures were characterized cytokinetically (flow cytometry/bromodeoxyuridine uptake) and immunocytochemically (static cytometry) for expression of CD14, CD11b (monocyte-macrophage) and DC-SIGN (dendritic cell: DCs) markers. RESULTS: Some treatments determined appearance of monocyte/macrophage, dendritic and apoptotic phenotypes, percentages of which were related to PMA dose used in step 1, and dependent on presence/absence of PMA and RA. PMA withdrawal induced dedifferentiation and partial restoration of proliferative activity, specially in 6 and 30 nm PMA-derived cells. Recovery in the presence of serum (fundamental to DC appearance) indicated that depending on differentiation level, cell proliferation and apoptosis were inversely correlated. Treatment with 30 nm PMA induced intermediate levels of monocytic-macrophagic differentiation, with expression of alternative means of differentiation and acquisition of DCs without using cytokines, after PMA withdrawal and RA stimulation. CONCLUSIONS: Our experimental conditions favoured differentiation, dedifferentiation and transdifferentiational pathways, in monocytic THP-1 cells, the balance of which could be related to both cell proliferation and cell death.

[Antiproliferative activity of cimetidine and a copper-cimetidine complex on rat fibroblasts cultured in vitro]. / Attività antiproliferativa della cimetidina e di un complesso rame-cimetidina in fibroblasti di ratto coltivati in vitro.

Cimetidine is an active H2-receptor antagonist which, combined with other drugs, has antitumor effect. It has been recently shown that some drugs complexed with copper increase their inhibitory effect on tumor growth and promote cell differentiation. In the present investigation we analysed the modifications of some cell proliferation parameters (mitotic, labelling and apoptotic indices and DNA synthesis) by microfluorometry and autoradiography in 2-day low and high cell density fibroblast cultures in presence of 20 microM cimetidine or copper-cimetidine complex. In low density cultures, a more intense proliferation activity was found while in the high density ones there was accumulation of 4c and 8c polyploid cells. The highest antiproliferative effect was found in low density cultures in presence of copper cimetidine complex, which caused mitotic anomalies and increased chromocenters. Simultaneous evaluation of nuclear DNA content and 3H-T labelling indicated the extension of G1 and G2 phases, followed by a lower cell proliferation and degeneration.

[Various nuclear and cytoplasmic parameters in relation to the evolution of an in vitro culture of rat fibroblasts]. / Studio di alcuni parametri nucleari e citoplasmatici in relazione all'evoluzione di una coltura in vitro di fibroblasti di ratto.

A cytokinetic study was performed on progressive subcultures of a cellular strain represented by fibroblasts (FG: Fibroblasts Galliera). Our previous studies on these cells showed morphofunctional changes in relation to cell ageing, indicating the stabilization of a cell line with high proliferative capacity. Furthermore, in the present work, we carried out a comparative analysis with a neoplastic strain (SGS/3A cells), characterized by a low state of cell differentiation. The morphological (fluorescence microscopy) and quantitative (flow cytometry) analysis, examined the relative DNA/protein and DNA/RNA ratios. Moreover, the replicative and mitotic activity and the apoptotic degeneration was determined. The results obtained indicated the existence in this biological model of a different relation between cell growth and cell proliferation. The analysis of our results allowed us to hypothesize that the cellular subpopulation selected during the culture progression of FG cells constitutes a cell line highly able of survival in vitro. In these cells the high proliferative activity is opposite to a reduced metabolism.

[Cell adhesion in neoplastic cells and syngeneic fibroblasts of the rat: effect of progesterone and estradiol]. / Adesione cellulare in cellule neoplastiche ed in fibroblasti singeneici di ratto: effetto del progesterone e dell'estradiolo.

Recent advances in the study on the importance of steroid receptors in the treatment of hormone-responsive tumors established the relationship between plasma membrane and the first steps of the cellular response to the endocrine stimulus. We have therefore found interesting to study the influence of estrogen and progesterone on cell adhesion of neoplastic (SGS/3A) and normal syngeneic (FG) cells. We demonstrated the presence of a nuclear estrogenic and a cytosolic progestin receptor in the SGS/3A cells. In syngeneic fibroblasts both receptors are absent. Cell adhesion kinetics obtained determining the percent of single labeled cells with 3H-leucine adhering to confluent monolayer (cell-cell adhesion) showed that the physiological concentrations of estradiol and progesterone induce an inhibition of cell-substratum adhesion (25-30%) in neoplastic cells, but do not influence cell-cell adhesion. In FG cells the two hormones cause an increase of cell-substratum adhesion (35-40%), but do not influence cell-cell adhesion. Results suggest that FG cells, although lacking cytoplasmic or nuclear receptors for estradiol and progesterone, probably have other steroid-receptive molecules involved in adhesive processes on their cell-surface.

Lymphocytes on sounding rocket flights.

NASA: Cell-cell interactions and the formation of cell aggregates are important events in the mitogen-induced lymphocyte activation. The fact that the formation of cell aggregates is only slightly reduced in microgravity suggests that cells are moving and interacting also in space, but direct evidence was still lacking. Here we report on two experiments carried out on a flight of the sounding rocket MAXUS 1B, launched in November 1992 from the base of Esrange in Sweden. The rocket reached the altitude of 716 km and provided 12.5 min of microgravity conditions.^ieng

Lymphocyte movements and interactions in microgravity.

NASA: Cell-cell contacts and the formation of aggregates play an important role in the mitogen induced in-vitro activation of lymphocytes. The fact that the formation of cell aggregates is only slightly reduced in microgravity suggests that cells are moving and interacting also in space. Direct evidence was obtained for the first time in an experiment performed on a sounding rocket flight, where the movements and interactions of free-floating, non activated cells could be observed in real time in microgravity. In an experiment performed on the IML-2 mission in July 1994, the movements and interactions of human lymphocytes during activation with the mitogen Con A were studied in real time in microgravity.^ieng

[Estrogen receptor levels in the liver of intact and gonadectomized rats and in cultured hepatocytes]. / Livelli recettoriali per gli estrogeni nel fegato di ratti intatti e gonadectomizzati ed in epatociti in coltura.

The concentrations of hepatic estrogen receptor were determined in intact and gonadectomized male and female rats. The hydroxylapatite assay demonstrated that the ablation of gonads induces, in the liver, an increase of estrogen receptors. The data obtained suggests that the synthesis of these receptors in the liver might be estrogen- and androgen-dependent. The same analysis performed on hepatocyte cytosol, derived from ovariectomized females, and cultured for 24, 48 and 72 hours, showed that time of culture is an important factor in determining a decrease of estrogen receptor concentrations in the cells. The results obtained allowed us to conclude that in the maintenance of the normal levels of the hepatic estrogen receptors, either sexual and non-sexual hormones are involved.