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The plasma membrane lipid composition in AH-130 hepatoma cells was found to change remarkably after polyenylphosphatidylcholine (PPC) treatment. Plasma membranes from cells grown in rats treated for 7 days i.v. with 20 mg/kg/day PPC, when compared to those of control cells, did not show significantly different amounts of cholesterol or phospholipids relative to protein content, but, surprisingly, the individual phospholipid distribution inside the two membrane leaflets changed dramatically. Phosphatidylcholine (PC), the major phospholipid in the external membrane leaflet, increased ~47% (p<0.001). By contrast, phosphatidylethanolamine (PE), the most important component of the inner leaflet, decreased nearly 37% (p<0.001), while sphingomyelin (SM) also decreased ~17%, (p=0.1). Tumor cells collected from control rats at the same time interval and observed by scanning electron microscopy, exhibited a spherical shape with numerous and randomly distributed long microvilli, the same morphological and ultrastructural features displayed by the implanted cells. Conversely, tumor cells from PPC-treated rats no longer showed the roundish cell profile, and microvilli appeared shortened and enlarged, with the formation of surface blebs. Transmission electron microscopy observations confirmed the morphological and ultrastructural cell changes, mainly seen as loss of microvilli and intense cytoplasmic vacuolization. Taken together, these results indicate that the new phospholipid class distribution in the plasma membrane leaflets, modifying tumor cell viable structures, produced heavy cell damage and in many cases brought about complete cellular disintegration.

Lauryl gallate is an antioxidant food additive showing low toxicity to normal cells. Here, its antiproliferative effect has been studied on three human breast cancer cell lines: estrogen-dependent, wild-type p53, MCF7; estrogen-independent, non-functional p53, MDA-MB-231 and MCF7 ADR, which overexpresses P-glycoprotein (P-gp) and displays a multidrug-resistant phenotype. Lauryl gallate inhibited proliferation and induced cell cycle alterations in all three cell lines without altering P-gp functionality in the drug-resistant cells. A stable arrest in G(1) phase was observed in MCF7, while a slow-down of cell cycle progression was induced in the other two cell lines. Lauryl gallate increased p53 expression only in MCF7, and upregulated p21(Cip1) and reduced cyclin D1 levels in all three cell lines. The induction of apoptosis, demonstrated by annexin V-FITC labeling, PARP cleavage and mitochondrial membrane depolarization and morphological alterations, were clearly detected in MCF7 ADR and MDA-MB-231 and to a minor extent in MCF7. Overexpression of Bcl-2 in MCF7 ADR cells demonstrated its protective role against morphological alterations and apoptosis. Lauryl gallate induction of p21(Cip1) and apoptosis observed in all three cell lines was regulated by Erk1/2 activation. These findings suggest a potential use of lauryl gallate against tumors harboring p53 mutations and drug-resistant phenotypes.

Hyperthermia is currently receiving widespread attention when associated with other therapeutic modalities, such as irradiation or chemotherapy, in the treatment of cancer. The occurrence of resistance to cytotoxic pharmacological agents in tumor cells, associated with several phenotypic alterations, is one of the major obstacles to successful anticancer chemotherapy. We investigated a new strategy to overcome multidrug resistance (MDR) cancer cells, using bovine serum amine oxidase (BSAO), which forms toxic products from spermine (H2O2 and aldehydes). The cytotoxicity of the products was evaluated in drug-sensitive (LoVo WT) and multidrug-resistant (LoVo DX) colon adenocarcinoma cells at 37 and 42 degrees C, using a clonogenic cell survival assay. Cytotoxicity was considerably enhanced at 42 degrees C. Both toxic species contributed to the thermal enhancement of cytotoxicity induced by BSAO and spermine. Cytotoxicity was eliminated in the presence of catalase and aldehyde dehydrogenase (ALDH). An interesting finding was that BSAO and spermine at <1 microM, which were non toxic at 37 degrees C, became cytotoxic at 42 degrees C and resemble thermosensitizers. Cell survival results and electron microscopy investigations suggest that, at 42 degrees C, LoVo DX cells are not resistant to the cytotoxic enzymatic oxidation products of spermine, as was already demonstrated in these cells at 37 degrees C. Moreover, microscopy modifications caused by both toxic products were more pronounced in LoVo DX than in LoVo WT cells, where morphological cytoplasmatic alterations were shown. Our findings suggest that hyperthermia combined with the enzymatic toxic oxidation products of spermine might be a promising anticancer strategy, mainly against MDR tumor cells.

The ability of Candida albicans to convert from the yeast (Y) form to mycelial forms through germ tube (GT) formation is considered a key feature of the transition of the organism from commensalism to virulence. We show here that human monocytes cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4 (IL-4) after phagocytosis of Y forms did not differentiate into dendritic cells (DCs); they retained CD14, did not acquire CD1a, and were unable to express the maturation markers CD83 and CCR7. Moreover, they did not produce IL-12p70 but secreted IL-10. In addition, they spontaneously expressed high levels of tumor necrosis factor alpha (TNF-alpha), IL-6, and IL-8 mRNA transcripts and were able to induce proliferation of alloreactive memory but not naïve T lymphocytes. Conversely, monocytes that had phagocytosed GT forms differentiated into mature CD83+ and CCR7+ DCs; however, there was no up-regulation of CD40, CD80, and major histocompatibility complex class II, irrespective of lipopolysaccharide (LPS) treatment. In addition, these cells were unable to produce IL-12 even after LPS stimulation, but they were not functionally exhausted, as shown by their capacity to express TNF-alpha and IL-8 mRNA transcripts. These cells were able to prime naïve T cells but not to induce their functional polarization into effector cells. These data indicate that phagocytosis of Y and GT forms has profound and distinct effects on the differentiation pathway of monocytes. Thus, the differentiation of human monocytes into DCs appears to be tunable and exploitable by C. albicans to elude immune surveillance.

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.

The occurrence of resistance to cytotoxic agents in tumor cells, associated with several phenotypic alterations, is one of the major obstacles to successful anticancer chemotherapy. A new strategy to overcome MDR of human cancer cells was studied, using BSAO, which generates cytotoxic products from spermine, H(2)O(2) and aldehyde(s). The involvement of these products in causing cytotoxicity was investigated in both drug-sensitive (LoVo WT) and drug-resistant (LoVo DX) colon adenocarcinoma cells. Evaluation of clonogenic cell survival showed that LoVo DX cells are more sensitive than LoVo WT cells. Fluorometric assay and treatments performed in the presence of catalase demonstrated that the cytotoxicity was due mainly to the presence of H(2)O(2). Cytotoxicity was eliminated in the presence of both catalase and ALDH. Transmission electron microscopic observations showed more pronounced mitochondrial modifications in drug-resistant than in drug-sensitive cells. Mitochondrial functionality studies performed by flow cytometry after JC-1 labeling revealed basal hyperpolarization of the mitochondrial membrane in LoVo DX cells. After treatment with BSAO and spermine, earlier and higher mitochondrial membrane depolarization was found in LoVo DX cells than in drug-sensitive cells. In addition, higher basal ROS production in LoVo DX cells than in drug-sensitive cells was detected by flow-cytometric analysis, suggesting increased mitochondrial activity in drug-resistant cells. Our results support the hypothesis that mitochondrial functionality affects the sensitivity of cells to the cytotoxic enzymatic oxidation products of spermine, which might be promising anticancer agents, mainly against drug-resistant tumor cells., (Copyright 2002 Wiley-Liss, Inc.)

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.

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.

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.

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.

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.

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.

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.

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.