Caspase-independent apoptosis is activated by diazepam-induced mitotic failure in HeLa cells, but not in human primary fibroblasts.

DZ, a benzodiazepine known to affect centrosome separation at prophase, leads to a higher degree of mitotic arrest in HeLa cells than in primary human fibroblasts. In fact, differently from fibroblasts, which undergo a transient block in prophase-to-prometaphase transition, a high proportion of tumor cells attempt to escape from the DZ-imposed mitotic block, fail to undergo complete mitosis and die by mitotic failure. DZ-treated samples showed certain biochemical hallmarks of apoptosis, such as induction of the proapototic Bax protein, mitochondrial alterations assessed by JC-1 staining and TEM analysis, PARP cleavage, and DNA fragmentation. However, in DZ-treated cells, we observed a very low or absent caspase activation as shown by immunofluorescence and immunoblot experiments with antibodies directed to activated caspases and by staining with the pancaspase inhibitor FITC-VAD-FMK. Experiments on mitochondrial depolymerization and apoptosis induction carried out in the presence of specific inhibitors of caspase-2 and caspase-3/7 indicated a caspase-independent apoptotic process induced by DZ. Accordingly, TEM analysis of treated cells revealed ultrastructural features resembling those reported for caspase-independent apoptosis. In conclusion, we hypothesize that HeLa cells override the prophase block imposed by DZ, producing a high rate of aberrant pro-metaphases, which, in turn, activates caspase-independent, apoptosis-like mitotic catastrophe.

Mitochondrial alterations induced by serum amine oxidase and spermine on human multidrug resistant tumor cells.

Multidrug resistance (MDR) has been studied extensively because it is one of major problems in cancer chemotherapy. The MDR phenotype is often due to overexpression of P-glycoprotein (P-gp), that acting as an energy-dependent drug efflux pump exports various anticancer drugs out of cells. The major goal of our investigation is to establish whether bovine serum amine oxidase (BSAO), which generates the products H(2)O(2) and aldehyde(s), from the polyamine spermine, is able to overcome MDR of human cancer cells. The cytotoxicity of the products was evaluated in both drug-sensitive (LoVo WT) and drug-resistant (LoVo DX) colon adenocarcinoma cells. A clonogenic cell survival assay demonstrated that LoVo DX cells were more sensitive than LoVo WT cells. Exogenous catalase protected cells against cytotoxicity mainly due to the formation of H(2)O(2). However, spermine-derived aldehyde(s) still induced some cytotoxicity. The cytotoxic effect was totally inhibited in the presence of both enzymes, catalase and NAD-dependent aldehyde dehydrogenase (ALDH). Transmission electron microscopy investigations showed that BSAO and spermine induced evident mitochondria alterations, more pronounced in MDR than in LoVo WT cells. The mitochondrial activity was checked by flow cytometry studies, labelling cells with the probe JC1, that displayed a basal hyperpolarized status of the mitochondria in multidrug-resistant cells. After treatment with amine oxidase in the presence of polyamine-spermine, the cells showed a marked increase in mitochondrial membrane depolarization higher in LoVo DX than in LoVo WT cells. Our findings suggest that toxic oxidation products formed from spermine and BSAO could be a powerful tool in the development of new anticancer treatments, mainly against MDR tumor cells.

Subcellular detection and localization of the drug transporter P-glycoprotein in cultured tumor cells.

In vitro studies on the cellular location of P-glycoprotein (Pgp) are reported with the aim to clarify the relationship between its intracellular expression and the multidrug resistance (MDR) level of tumor cells. Pgp was found abnormally expressed on the plasma membrane of tumor cells with "classical" MDR phenotype. However, Pgp was also often detected on the nuclear envelope and on the membrane of cytoplasmic organelles. The hypothesis that this drug pump maintains a transport function when located in these compartments, is still under debating. Our results, together with those obtained by other researchers, demonstrate that cytoplasmic Pgp regulates the intracellular traffic of drugs so that they are no more able to reach their cellular targets. In particular, we revealed that in MDR breast cancer cells (MCF-7) a significant level of Pgp was expressed in the Golgi apparatus. A similar result was found in human melanoma cell lines, which never undergone cytotoxic drug treatment and did not express the transporter molecule on the plasma membrane. A strict relationship between intracellular Pgp and intrinsic resistance was demonstrated in a human colon carcinoma (LoVo) clone, which did not express the drug transporter on the plasma membrane. Finally, a structural and functional association between Pgp and ERM proteins has been discovered in drug-resistant human T- lymphobastoid cells (CEM-VBL 100). Our findings strongly suggest a pivotal role of the intracytoplasmic Pgp in the transport of drugs into cytoplasmic vesicles, thus actively contributing to their sequestration and transport outwards the cells. Thus, intracellular Pgp seems to represent a complementary protective mechanism of tumor cells against cytotoxic agents.

Saposin D solubilizes anionic phospholipid-containing membranes.

Saposin (Sap) D is a late endosomal/lysosomal small protein, generated together with three other similar proteins, Sap A, B, and C, from the common precursor, prosaposin. Although the functions of saposins such as Sap B and C are well known (Sap B promotes the hydrolysis of sulfatides and Sap C that of glucosylceramide), neither the physiological function nor the mechanism of action of Sap D are yet fully understood. We previously found that a dramatic increase of Sap D superficial hydrophobicity, occurring at the low pH values characteristic of the late endosomal/lysosomal environment, triggers the interaction of the saposin with anionic phospholipid-containing vesicles. We have presently found that, upon lipid binding, Sap D solubilizes the membranes, as shown by the clearance of the vesicles turbidity. The results of gel filtration, density gradient centrifugation, and negative staining electron microscopy demonstrate that this effect is due to the transformation of large vesicles to smaller particles. The solubilizing effect of Sap D is highly dependent on pH, the lipid/saposin ratio, and the presence of anionic phospholipids; small variations in each of these conditions markedly influences the activity of Sap D. The present study documents the interaction of Sap D with membranes as a complex process. Anionic phospholipids attract Sap D from the medium; when the concentration of the saposin on the lipid surface reaches a critical value, the membrane breaks down into recombinant small particles enriched in anionic phospholipids. Our results suggest that the role played by Sap D is more general than promoting sphingolipid degradation, e.g. the saposin might also be a key mediator of the solubilization of intralysosomal/late endosomal anionic phospholipid-containing membranes.

Morphological and functional alterations of human erythrocytes induced by SiO2 particles: An electron microscopy and dielectric spectroscopy study.

The interaction of aerosil particles with human erythrocytes was investigated by electron microscopy methods complemented with hemolysis and radio wave dielectric spectroscopy to elucidate the extent of morphological and functional modification induced by aerosil surface. Scanning electron microscopy and freeze-fracturing techniques were used to follow morphological and ultrastructural modifications and hemolysis tests and radio wave dielectric spectroscopy to monitor the membrane damage. All experimental results indicate that there is an effect depending on both silica concentration and incubation time. Our results are in good agreement with an interaction model based on membrane protein denaturation due to the electrostatic attraction between (-SiO-) groups at the silica surface and proteins embedded in the membrane. The process is time-limited and reaches saturation after about 20 min. The extent of the damage is determined mainly by the ratio between cell and aerosil surface, that is, aerosil concentration. Limited damage is observed, especially when little aerosil surface per cell is available. Conversely, strong membrane damage is obtained when aerosil surface is considerable. In any case, due to the high surface/volume of aerosil particles used in our experiments we obtained considerable membrane damage with small weight concentrations.

Interaction between human interleukin-2-activated natural killer cells and heat-killed germ tube forms of Candida albicans.

Human interleukin-2-activated natural killer (LAK) cells are able to recognize and to bind to both live and heat-killed germ tube forms of Candida albicans, establishing a wide and intimate contact as revealed by electron microscopic observations. Following the interaction, LAK cells are activated: an increased expression of some cytokine mRNA (in particular, TNF-alpha, GM-CSF, and IFN-gamma) has been revealed by RT-PCR and perforin secretion has been suggested by immunofluorescence microscopy. Nonetheless, neither morphological damage or growth inhibition of fungal target cells have been detected. Instead, evident signs of cell damage could be noticed in interacting LAK cells. Moreover, the observation by transmission electron microscopy of LAK cell-germ tube conjugates revealed the presence of apoptotic cells. The analysis of LAK cell cytotoxic activity against DAUDI cells showed that the lymphocytic effector underwent a significant reduction in its lytic capability after the interaction with C. albicans. The results obtained in this in vitro study seem to indicate that in such an interaction LAK cells cannot directly inhibit or kill the fungal pathogen by using their lytic machinery but they secrete those cytokines which have stimulatory effects on phagocytic cells. The ultimate results are the programmed death of LAK cells and the enhancement of the fungicidal activity exerted by competent cells.

Expression of the complement-binding protein (MP60) of Candida albicans in experimental vaginitis.

The expression of the Candida albicans complement-binding C3d protein (MP60) was investigated both in vitro and in vivo by immunogold labelling and electron microscopy. In vivo expression was determined in a rat vaginitis model. Reactivity of in vitro-grown cells to an anti-MP60 rabbit serum was associated with both cytoplasmic and cell wall sites. Immunostaining in the cell wall of both yeast and hyphae was most concentrated in the inner, electron-lucid layer. Immunogold stained preparations of C. albicans from vaginal smears of infected animals also showed intense localization of the MP60 in the inner cell wall, plasma membrane. However, immunogold label was also intense at the cell surface in these samples, mostly in the area of close adherence with the keratinocytes of the vaginal epithelia. These observations indicate that MP60 is expressed both in vitro and in vivo, but to a different degree in the different cell wall layers.

Ultrastructural localization of the secretory aspartyl proteinase in Candida albicans cell wall in vitro and in experimentally infected rat vagina.

Detection and ultrastructural localization of aspartyl proteinase (Sap) in Candida albicans experimentally infecting rat vagina were studied. Two Sap-positive (Sap+) and one Sap-negative (Sap-) strains of the fungus, endowed with high and low experimental vaginopathic potential, respectively, were used. Both Sap+ strains produced consistent Sap levels in the rat vagina, while the Sap- strain did not produce any measurable Sap. Electron microscopy of thin sections of chemically-fixed vaginal scrapings showed clear evidence of hyphae of proteolitic strains of C. albicans invading the keratinized epithelial cell layer of the vagina. The fungal cells exhibited a pronounced fibrillar layer on the cell wall with a marked intermixing of fungal and vaginal materials especially pronounced at the hyphal tip. Post-embedding immunogold techniques with the use of anti-Sap polyclonal and the specifically generated monoclonal antibody GF1 showed that Sap was essentially localized in the cell wall of C. albicans early during infection, in a cytological pattern mirroring Sap localization in C. albicans cells grown in Sap-inductive media in vitro. In summary, the data offer a new biochemical and ultrastructural evidence that Sap is actively secreted during experimental rat vaginitis by C. albicans. Cell wall localization of Sap is probably inherent to this active secretion process.

Noninhibitory binding of human interleukin-2-activated natural killer cells to the germ tube forms of Candida albicans.

During incubation in vitro with yeast or germ tube forms of Candida albicans, only 2 to 6% of freshly isolated human natural killer (NK) cells (> 85% CD16+, CD56+, CD3-; < 15% CD3+; cytolytic for the NK-susceptible target K562 but not for the NK-resistant target DAUDI), were seen to interact with the fungal cells. As seen under the electron microscope, the contact area had a limited extent and was narrow, and neither the surface nor the intracytoplasmic organization of the NK cell was altered. In contrast, more than 30% of interleukin-2-activated NK (LAK) cells (> 96% CD16+, CD56+, CD3-; 1.5% CD3+; cytolytic for both K562 and DAUDI targets) interacted closely with the fungus. This interaction was particularly extensive with the surface of the fungal germ tube that was intimately enveloped by villous protrusions from the lymphocyte surface. The fungus-interacting LAK cell also showed a remarkable redistribution of surface microvilli and polarization of cytoplasmic organelles, such as the Golgi apparatus, centrioles, and granules, toward the area of fungal contact. Together with the elevated cytolytic potential against the K562 and DAUDI targets, all the morphological data suggested the presence of a potentially active lytic machinery in the fungus-interacting LAK cell. Nonetheless, two independent assays for anticandidal activity did not show consistent killing or fungal growth inhibition by either fresh NK or LAK cells. While offering direct evidence of the strong interaction between human LAK cells and the germ tubes, precursors of tissue-invasive hyphal forms of C. albicans, our observations also suggest that this interaction may not be sufficient to kill the fungus or arrest its growth.

Differential cell surface expression of mannoprotein epitopes in yeast and mycelial forms of Candida albicans.

The ultrastructural localization of mannoprotein constituents (MP) of the cell wall of yeast and hyphal forms of Candida albicans was studied by immunoelectron microscopy. To this aim, two monoclonal antibodies (mAbs AF1 and 1D10), recognizing distinct oligomannoside epitopes of MP molecules, and a second antibody coupled to colloidal gold, were employed. Preembedding methods revealed the presence of both AF1- and, albeit to a lesser intensity, 1D10-epitopes within the fibrillar, capsular layer of yeast cells of the fungus, provided this capsule was preserved and stabilized by treatment of whole cells with Concanavalin A. These cell surface-associated MP were absent in hyphal cells, despite the presence in these cells of a capsular layer not different in form and thickness from that present in yeast cells. Postembedding methods showed that both yeast and hyphal forms of growth of C. albicans synthesized the relevant mannoproteins and similarly incorporated them into inner layers of the cell wall. Apparently, however, the "export" of these MP to the outermost, capsular layer occurred in yeast but not in hyphal cells. These ultrastructural data, coupled with previous biochemical ones, emphasize form-associated patterns of MP expression on Candida cell surface. Given the value of MP as main immunogenic components of Candida, this differential expression could be a means by which the fungus evades from, or attenuates host's response.

Evaluation of an experimental periodontal ligament for dental implants.

To improve the devices used in dental implantology, a new experimental supporting element has been developed. The device, made of titanium, poly(methylmethacrylate) and Dacron tissue, has been tested for the osteointegrability of its outer Dacron coat by implants in mandibular bone of rabbits. The Dacron filamentous tissue became incorporated by the bone at 3 month post-implantation. This might provide a reliable mechanical anchorage of the device and a barrier against epithelial proliferation and microbial contamination. Preliminary results relating to in vitro evaluation of the poly(methyl methacrylate)-Dacron fixation strength predict favourable mechanical behaviour at this interface and in the complete implanted device.

Effects of daunomycin on the microtubular network: a cytochemical study on a human melanoma cell line.

The interaction of daunomycin (DAU), an anthracyclinic antibiotic employed as antitumoral agent, with microtubules, has been investigated by cytochemical and morphological methods on a human melanoma cell line (H14). Results obtained indicated that DAU was able to modulate the microtubule reassembly in cells treated with colcemid; such an effect proved to be dose-dependent. In particular, it has been observed that a low dose of DAU (0.05 microM) seemed to favor the microtubule reassembly whereas a higher dose (0.10 microM) impaired this process. In addition, when the anthracyclinic antibiotic was employed together with colcemid, both the cell detachment and the depolymerization of microtubules induced by the mitotic poison were hampered. These effects were dose-dependent and were better accomplished when DAU was used at an equimolar or at higher dose than that employed for the antimicrotubular agent. Moreover, the treatment of cells with DAU alone induced the stabilization of the microtubules, making them more resistant to the action of antimicrotubular agents. This effect could in part explain the antagonistic action exerted by DAU against colcemid. These observations seem to confirm that the microtubular network is an important target involved in the mechanism of action of the anthracyclinic antibiotics.

Suicide behavior of target cells after binding with natural killer cells.

Human natural killer (NK) cell activity seems to be related to the integrity and function of the cytoskeletal apparatus. It has been hypothesized that microfilaments and microtubules play a pivotal role. In particular, the binding of the NK cell to the target cell requires microfilament integrity, and the lysis of bound targets seems to depend on microtubule assembly. We focused on the changes occurring in cytoskeletal elements and surface structures of NK cells and of target cells highly sensitive to NK activity (K562). Our observations, performed by fluorescence and scanning electron microscopy, besides confirming a rearrangement of the cytoskeletal apparatus in the effector cell, provide evidence that target cell cytoskeletal elements are involved in NK cell function. In K562 cells, after binding with NK cells, there is marginal rearrangement of actin and polarization of tubulin and vimentin in the contact regions, accompanied by modification of surface structures. These findings suggest that the target cell plays an active role in its own death by participating in the formation of an extended area of intimate contact with the killer cell. In addition, they lend credence to the surprising proposal that NK cells may induce a suicide mechanism in target cells.

Interaction of anthracyclinic antibiotics with cytoskeletal components of cultured carcinoma cells (CG5).

The effects of doxorubicin (adriamycin, ADR) and daunorubicin (daunomycin, DAU), two anthracyclinic antibiotics, on a human breast carcinoma cell line (CG5) were studied by cytochemical and morphological methods. Both ADR and DAU were capable of inducing the multinucleation and spreading phenomena, associated with a decrease of the cell growth rate. DAU appeared to be more effective than ADR at the tested concentrations (10(-5), 5 x 10(-5) mM), in affecting the cell growth as well as in inducing multinucleation. As revealed by scanning electron microscopy, spreading and multinucleation were accompanied by a remarkable redistribution of surface structures. Moreover, a dose- and time-dependent rearrangement of the underlying cytoskeletal components was clearly detected. In addition, both ADR and DAU at 5 x 10(-5) mM seemed to favor the rebuilding of microtubules after treatment with colcemid, while a higher dose (10(-4) mM) exerted the opposite effect. Furthermore, both anthracyclines prevented the action of the antimicrotubular agent. When recovered after treatment with cytochalasin B, in presence of ADR (or DAU) (5 x 10(-5), 10(-4) mM), cells showed a microfilament pattern rearranged differently as compared to that of cells recovered in anthracycline-free medium. The results reported here strongly suggest the involvement of actin and tubulin in CG5 cell response to ADR and DAU treatments. Thus, the cytoskeletal apparatus is confirmed as another target involved in the mechanism of action of anthracyclines.

Adriamycin-plasma membrane interaction in human erythrocytes.

A great body of data increasingly point to the cell membrane as an important target for adriamycin (ADR). However, the exact mechanism by which ADR exerts its cytotoxic action through the interaction with the plasma membrane is still unknown. In this study, the interaction of ADR with red blood cells from healthy donors was investigated by freeze-fracturing (FF) and scanning electron microscopy (SEM). The results obtained can be summarized as follows: a) a dose-dependent modification in the intramembrane particle (IMP) distribution was revealed by FF on both fracture faces of the plasma membrane of erythrocytes treated with 50 or 100 microM ADR; b) SEM observations allowed to reveal a discocyte-stomatocyte transition induced by 50 microM ADR and the formation of mottled cells at the higher dose; c) these morphological and ultrastructural changes were not related to lipid peroxidation as demonstrated by experiments with radical scavengers or strong oxidant substances; d) the analysis of IMP density seemed to rule out a segregation process of membrane proteins suggesting that ADR interacts with the plasma membrane by becoming incorporated within the lipid bilayer.

Effects of glutaraldehyde and glycerol on freeze-fractured Escherichia coli.

Glutaraldehyde and glycerol are widely used in the freeze-fracture technique as sample pretreatments before rapid freezing. However, they can both introduce relevant structural changes and influence the visualization of the fracture faces and surfaces of membranes. A comparison of the results obtained on E. coli cells differently pretreated with glutaraldehyde and glycerol is presented. In particular the effect on the distribution and density of the intramembranous particles (IMP) is pointed out. Glycerol treatment at 310 K introduces an IMP redistribution, outlined by the appearance of several smooth areas on the fracture faces of the cytoplasmic membrane, which is prevented by glutaraldehyde prefixation at the same temperature. On the other hand, glutaraldehyde treatment at 310 K following glycerol incubation results in the disappearance of the smooth areas, suggesting a substantial change in the IMP distribution caused by the fixative. Cells shifted down to 277 K and treated with glycerol at this temperature before quick-freezing, present on the convex fracture face of the cytoplasmic membrane large smooth areas resulting from a lipid transition while the smaller areas observed at 310 K are not detectable. Glutaraldehyde treatment at 277 K seems also to be responsible for a redistribution of IMP since poorly delimited large smooth areas, containing several IMP, can be observed. In this paper the results of a statistical analysis are also reported, showing that the IMP density can be strongly influenced by pretreatments.

Electron microscope identification of mesosomes in some water strains of Leptospira by reduction of tetrazolium chloride to formazans.

Rounded granules protruding from the external surface of some water Leptospira strains and considered as mesosomes were studied by electron microscope and by means of the intravital reduction of tetrazolium chloride. Satisfactory results were obtained with two of the strains studied. It appears that neither the culture medium nor the age of the culture have any influence on the unusual position of the mesosomes.