Autophagy is induced by adenoviral-mediated interferon alpha treatment in interferon resistant bladder cancer and normal urothelial cells as a cell death protective mechanism but not by the bystander factors produced.

We have previously shown that adenoviral-mediated interferon alpha (Ad-IFNalpha) treatment is highly cytotoxic to tumor cells which are resistant to the IFNalpha protein. We now report that autophagy is produced after Ad-IFNalpha treatment of either IFN resistant bladder cancer cells (UC9 and KU7) or the normal urothelial cell line (TERT-NHUC). After Ad-IFNalpha infection autophagosomes, an early stage of autophagy, were seen in cancer cells whereas autophagolysosomes, a later stage of autophagy, were observed mostly in normal cells by electron microscopy. Conditioned medium from either normal or bladder cancer cells obtained after Ad-IFNalpha infection, however, produced no autophagy when placed on the bladder cancer cells, although again marked cytotoxicity was observed. This indicated that the autophagy seen was related to the direct effect of Ad-IFNalpha transfection and expression rather than to the bystander factors produced. In addition, autophagic changes were seen using LysoTracker Red DND-99 in both normal and cancer cells. We also documented that Ad-IFNalpha treatment produces the autophagic protein form, light chain 3 (LC3)-II, in cancer cells but not normal cells, which in turn was inhibited by the autophagic inhibitor, 3-methyladenine (3-MA). This inhibition of autophagy resulted in a significant increase in apoptotic cell death as measured by the sub-G1 population. We hypothesize that the autophagy seen in normal urothelial cells is a protective response and is allowed to be completed, providing a survival mechanism after Ad-IFN treatment, whereas the autophagy produced in IFN resistant cancer cells is not allowed to be completed and is insufficient to significantly suppress cytotoxicity.

Vital function of PRELI and essential requirement of its LEA motif.

Proteins containing the late embryogenesis abundant (LEA) motif comprise a conserved family, postulated to act as cell protectors. However, their function and mechanisms of action remain unclear. Here we show that PRELI, a mammalian LEA-containing homolog of yeast Ups1p, can associate with dynamin-like GTPase Optic Atrophy-1 (OPA1) and contribute to the maintenance of mitochondrial morphology. Accordingly, PRELI can uphold mitochondrial membrane potential (ΔΨ(m)) and enhance respiratory chain (RC) function, shown by its capacity to induce complex-I/NADH dehydrogenase and ATP synthase expression, increase oxygen consumption and reduce reactive oxygen species (ROS) production. PRELI can also inhibit cell death induced by STS, TNF-α or UV irradiation. Moreover, in vitro and in vivo dominant-negative overexpression of mutant PRELI/LEA(-) (lacking the LEA motif) and transient in vitro PRELI-specific knockdown can render lymphocytes vulnerable to apoptosis, cause mouse embryo lethality and revert the resistance of lymphoma cells to induced death. Collectively, these data support the long-presumed notion of LEA protein-dependent mechanisms of cytoprotection and suggest that PRELI interacts with OPA1 to maintain mitochondria structures intact, sustain balanced ion(-)/proton(+) gradients, promote oxidative phosphorylation reactions, regulate pro- and antiapoptotic protein traffic and enable cell responses to induced death. These findings may help to understand how bioenergetics is mechanistically connected with cell survival cues.

Proteasome inhibitors activate autophagy as a cytoprotective response in human prostate cancer cells.

The ubiquitin-proteasome and lysosome-autophagy pathways are the two major intracellular protein degradation systems that work cooperatively to maintain homeostasis. Proteasome inhibitors (PIs) have clinical activity in hematological tumors, and inhibitors of autophagy are also being evaluated as potential antitumor therapies. In this study, we found that chemical PIs and small interfering RNA-mediated knockdown of the proteasome's enzymatic subunits promoted autophagosome formation, stimulated autophagic flux, and upregulated expression of the autophagy-specific genes (ATGs) (ATG5 and ATG7) in some human prostate cancer cells and immortalized mouse embryonic fibroblasts (MEFs). Upregulation of ATG5 and ATG7 only occurred in cells displaying PI-induced phosphorylation of the eukaryotic translation initiation factor 2 alpha (eIF2alpha), an important component of the unfolded protein responses. Furthermore, PIs did not induce autophagy or upregulate ATG5 in MEFs expressing a phosphorylation-deficient mutant form of eIF2alpha. Combined inhibition of autophagy and the proteasome induced an accumulation of intracellular protein aggregates reminiscent of neuronal inclusion bodies and caused more cancer cell death than blocking either degradation pathway alone. Overall, our data show that proteasome inhibition activates autophagy through a phospho-eIF2alpha-dependent mechanism to eliminate protein aggregates and alleviate proteotoxic stress.

Increased mitochondrial biogenesis in primary leukemia cells: the role of endogenous nitric oxide and impact on sensitivity to fludarabine.

B cell chronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia in the Western hemisphere, yet many biological and molecular features of the disease remain undefined. CLL cells generate increased levels of radical species such as superoxide and nitric oxide (NO), which is associated with mitochondrial DNA mutations. Considering that NO levels can affect mitochondrial biogenesis, we hypothesized that the inherent nitrosative stress in CLL cells may lead to hyperactive mitochondrial biogenesis. Here we report that primary CLL cells contained significantly more mitochondria than normal lymphocytes and that their mitochondrial mass was significantly related to endogenous NO levels. Expression of the mitochondrial biogenesis factors nuclear respiratory factor-1 and mitochondrial transcription factor A was elevated in most CLL specimens examined and appeared to be related to cellular NO levels. Treatment of B cells with exogenous NO caused a substantial increase in mitochondrial mass. In vitro sensitivity of CLL cells to fludarabine was highly related to mitochondrial mass in that cells with greater mitochondrial mass were less sensitive to the drug. Taken together, our results suggest that NO is a key mediator of mitochondrial biogenesis in CLL and that modulation of mitochondrial biogenesis by NO may alter cellular sensitivity to fludarabine.

Topical treatment with liposomes containing T4 endonuclease V protects human skin in vivo from ultraviolet-induced upregulation of interleukin-10 and tumor necrosis factor-alpha.

Exposing human skin to ultraviolet radiation causes DNA damage, sunburn, immune alterations, and eventually, skin cancer. We wished to determine whether liposomes containing a DNA repair enzyme could prevent any of the acute effects of irradiation when applied after ultraviolet exposure. Fifteen human patients with a prior history of skin cancer were exposed to two minimal erythema doses of ultraviolet radiation on their buttock skin. Liposomes containing T4 endonuclease V or heat-inactivated enzyme were applied immediately and at 2, 4, and 5 h after ultraviolet irradiation. Transmission electron microscopy after anti-T4 endonuclease V-staining and immunogold labeling on biopsies taken at 6 h after ultraviolet exposure revealed that the enzyme was present within cells in the skin. Immunohistochemical DNA damage studies suggested a trend toward improved DNA repair at the active T4 endonuclease V liposome-treated test sites. Although the active T4 endonuclease V liposomes did not significantly affect the ultraviolet-induced erythema response and microscopic sunburn cell formation, they nearly completely prevented ultraviolet-induced upregulation of interleukin-10 and tumor necrosis factor-alpha RNA message and of interleukin-10 protein. These studies demonstrate that liposomes can be used for topical intracellular delivery of small proteins to human skin and suggest that liposomes containing DNA repair enzymes may provide a new avenue for photoprotection against some forms of ultraviolet-induced skin damage.

Phenotypic and ultrastructural properties of antigen-presenting cells involved in contact sensitization of normal and UV-irradiated mice.

We investigated the surface phenotype and localization of hapten in antigen-presenting cells involved in the induction of contact hypersensitivity or tolerance. Dendritic cells collected 18 h earlier from the draining lymph nodes of mice sensitized with fluorescein isothiocyanate (FITC), which induce contact hypersensitivity upon injection into the foot-pads of naive mice, stimulated proliferation of lymphocytes from FITC-specific T-cell lines. By immunoelectron microscopy, these cells expressed high amounts of surface Ia molecules but had a negligible amount of FITC on the cell membrane. The majority of the FITC was localized in discrete structures in the cytoplasm, including mitochondria, endocytic vesicles, lysosomes, and cored tubules. In contrast, lymph node cells conjugated with FITC in vitro did not stimulate proliferation of FITC-specific T cells and showed a heavy, uniform distribution of FITC throughout the cytoplasm. Draining lymph node cells from mice exposed to ultraviolet (UV) radiation and then sensitized by applying FITC to the UV-irradiated skin, which induce tolerance upon injection into naive mice, induced significantly less proliferation of FITC-specific T cells than draining lymph node cells from unirradiated mice. The differences in activity of these cells, relative to draining lymph node cells from unirradiated, FITC-sensitized mice could not be attributed to a decreased number of Ia+ dendritic cells in the DLN, decreased surface expression of Ia molecules on these cells, or an alteration in the intracellular localization of hapten. However, a significantly higher percentage of the FITC+ dendritic cells from UV-irradiated mice expressed mac-1, -2, and -3 and F4/80 macrophage markers than did those from unirradiated animals, and fewer cells contained Birbeck granules, suggesting that a different population of Ia+ antigen-presenting cells may reach the draining lymph nodes of UV-irradiated mice.

The pattern of food hypersensitivity in patients with onset after 10 years of age.

One hundred and twelve patients with a history of immediate adverse reaction after food ingestion and positive skin test to food are presented with the result of food challenge. In all patients the symptoms started after 10 years of age; most presented with recurrent short-lasting urticarial rash, often accompanied by rhinitis. In the majority of the patients, skin tests were positive to multiple food allergens, but 67% of these responded to oral provocation by only one allergen. One-third of the patients had a history of allergic symptoms following exercise after meals, but in only one-third of these symptoms were reproducible in the laboratory. Fruit and vegetables were the main allergens responsible for food hypersensitivity. Food allergy can develop after the first 10 years of life. Fruit and vegetables are the main cause of food allergy in these patients, while milk and egg are the least common. These findings differ from those in early childhood where milk and eggs are the main allergens.

Characteristics of antigen-presenting cells involved in contact sensitization of normal and UV-irradiated mice.

Exposure of mice to ultraviolet (UV)-B (280-320 nm) radiation alters their immune response to contact-sensitizing haptens applied to UV-irradiated skin. Under these conditions, the contact-hypersensitivity response is reduced, and hapten-specific suppressor T lymphocytes can be found in the spleen. Considerable evidence suggests that the epidermal Langerhans cell is one of the principal targets of this effect of UV irradiation that leads, ultimately, to suppressor cell formation. We are using a combination of cell-surface markers and light and electron microscopy to determine whether differences can be detected in the antigen-presenting cells involved in contact sensitization of normal and UV-irradiated mice.

Internalization of Ia molecules into Birbeck granule-like structures in murine dendritic cells.

Dendritic cells isolated from the draining lymph nodes of mice sensitized epicutaneously with hapten are potent antigen-presenting cells and contain Birbeck granules and cored tubules characteristic of antigen-activated epidermal Langerhans cells. We used immunogold labeling and transmission electron microscopy to follow the internalization of Ia molecules in these antigen-presenting cells. We found that Ia molecules were internalized into Birbeck granule-like structures in the antigen-activated dendritic cells. Computer reconstruction of serial sections of the dendritic cells demonstrated that these structures span the cytoplasm from the cell membrane to the nuclear membrane and are associated with lysosomes. The internalization of Ia molecules into these structures supports the hypothesis that the Birbeck granule-like structures are derived from the cell membrane and are involved in the antigen-processing/presenting function of the dendritic cells.

Immunohistochemistry and histochemistry of the inner ear gelatinous membranes and statoconia of the chick (Gallus domesticus).

This study was undertaken to investigate whether the gelatinous membranes (GMs) of the chick (Gallus domesticus) inner ear contain glycoproteins and proteoglycans, as is the case in mammals, and whether or not the relative concentrations of glycoproteins and proteoglycans change during development. We used electron-microscopic histochemistry with tannic acid, Alcian blue and ruthenium red and immunoperoxidase with monoclonal antibodies to fibronectin and keratan sulfate. Both techniques were applied to material from newly hatched chicks in various developmental stages. In hatchlings, tannic acid, which precipitates mainly proteoglycans, stained the mineralizing GMs of the utricle, saccule and lagenar macula darker than it did the nonmineralizing GMs of the cristae and tectorial membrane of the basilar papilla, while Alcian blue and ruthenium red stained all GMs in the vestibule and basilar papilla almost to the same degree. Antikeratan sulfate (a proteoglycan marker) stained the GMs intensively, while antifibronectin (glycoprotein monoclonal marker) stained them less intensively. In the mineralizing GMs of the embryos, the immunoreactivity (density) of keratan sulfate almost doubles between days 7 (stages 31) and 21 (stage 46), while the density of fibronectin does not seem to change as much. Different concentrations of glycoproteins and proteoglycans in each GM may impart to it the capacity to remain unmineralized, as is the case for cupulae of the vestibule and tectorial membrane of the basilar papilla, or mineralized, as is the case for the statoconial membrane of the maculae.