The DNA damage checkpoint precedes activation of ARF in response to escalating oncogenic stress during tumorigenesis.

Oncogenic stimuli trigger the DNA damage response (DDR) and induction of the alternative reading frame (ARF) tumor suppressor, both of which can activate the p53 pathway and provide intrinsic barriers to tumor progression. However, the respective timeframes and signal thresholds for ARF induction and DDR activation during tumorigenesis remain elusive. Here, these issues were addressed by analyses of mouse models of urinary bladder, colon, pancreatic and skin premalignant and malignant lesions. Consistently, ARF expression occurred at a later stage of tumor progression than activation of the DDR or p16(INK4A), a tumor-suppressor gene overlapping with ARF. Analogous results were obtained in several human clinical settings, including early and progressive lesions of the urinary bladder, head and neck, skin and pancreas. Mechanistic analyses of epithelial and fibroblast cell models exposed to various oncogenes showed that the delayed upregulation of ARF reflected a requirement for a higher, transcriptionally based threshold of oncogenic stress, elicited by at least two oncogenic 'hits', compared with lower activation threshold for DDR. We propose that relative to DDR activation, ARF provides a complementary and delayed barrier to tumor development, responding to more robust stimuli of escalating oncogenic overload.

Molecular effects of advanced glycation end products on cell signalling pathways, ageing and pathophysiology.

Advanced glycation end-products (AGEs) are a heterogeneous group of compounds formed by the Maillard chemical process of non- enzymatic glycation of free amino groups of proteins, lipids and nucleic acids. This chemical modification of biomolecules is triggered by endogeneous hyperglycaemic or oxidative stress-related processes. Additionally, AGEs can derive from exogenous, mostly diet-related, sources. Considering that AGE accumulation in tissues correlates with ageing and is a hallmark in several age-related diseases it is not surprising that the role of AGEs in ageing and pathology has become increasingly evident. The receptor for AGEs (RAGE) is a single transmembrane protein being expressed in a wide variety of human cells. RAGE binds a broad repertoire of extracellular ligands and mediates responses to stress conditions by activating multiple signal transduction pathways being mostly responsible for acute and/or chronic inflammation. RAGE activation has been implicated in ageing as well as in a number of age-related diseases, including atherosclerosis, neurodegeneration, arthritis, stoke, diabetes and cancer. Here we present a synopsis of findings that relate to AGEs-reported implication in cell signalling pathways and ageing, as well as in pathology. Potential implications and opportunities for translational research and the development of new therapies are also discussed.

Specific lipofuscin staining as a novel biomarker to detect replicative and stress-induced senescence. A method applicable in cryo-preserved and archival tissues.

There is shortage of extensive clinicopathologic studies of cellular senescence because the most reliable senescence biomarker, the detection of Senescence-Associated-beta-galactosidase activity (SA-ß-gal), is inapplicable in archival material and requires snap-frozen tissues. We validated the histochemical Sudan-Black-B (SBB) specific stain of lipofuscin, an aggregate of oxidized proteins, lipids and metals, known to accumulate in aged tissues, as an additional reliable approach to detect senescent cells independently of sample preparation. We analyzed cellular systems in which senescence was triggered by replicative exhaustion or stressful stimuli, conditional knock-in mice producing precancerous lesions exhibiting senescence, and human preneoplastic lesions known to contain senescent cells. In the above settings we demonstrated co-localization of lipofuscin and SA-ß-gal in senescent cells in vitro and in vivo (cryo-preserved tissue), strongly supporting the candidacy of lipofuscin for a biomarker of cellular senescence. Furthermore, cryo-preserved tissues positive for SA-ß-gal were formalin-fixed, paraffin-embedded, and stained with SBB. The corresponding SA-ß-gal positive tissue areas stained specifically for lipofuscin by SBB, whereas tissues negative for SA-ß-gal were lipofuscin negative, validating the sensitivity and specificity of the SBB staining to visualize senescent cells in archival material. The latter unique property of SBB could be exploited in research on widely available retrospective tissue material.

Phorbol myristate acetate induces changes on F-actin and vinculin content in immature rat Sertoli cells.

Actin and vinculin are two of the most abundant cytoskeletal proteins, widely expressed in nearly all types of eukaryotic cells. It has been well established that long-term exposure to the tumor promoter phorbol myristate acetate (PMA) affects Sertoli cell morphology, as well as F-actin and vinculin organization in vitro. To analyze in a quantitative manner the F-actin and vinculin content of rat immature Sertoli cells in vitro in response to PMA exposure, cytoskeletal fractions were prepared following extraction with Triton X-100. Analysis of the isolated cytoskeletal fractions by immunoblotting showed that exposure of immature rat Sertoli cells to PMA for 8h has an appreciable effect on the cellular level of both the actin and vinculin. Interestingly, as revealed by using calphostin C, a specific protein kinase C inhibitor, the observed F-actin and vinculin changes are most probably mediated by a mechanism that depends on protein kinase activity. A discussion is made concerning PKC modulation by PMA and the subsequent actin and vinculin quantitative changes and reorganization, phenomena that have been closely related to cell transformation.

Ageing research in Greece.

Ageing research in Greece is well established. Research groups located in universities, research institutes or public hospitals are studying various and complementary aspects of ageing. These research activities include (a) functional analysis of Clusterin/Apolipoprotein J, studies in healthy centenarians and work on protein degradation and the role of proteasome during senescence at the National Hellenic Research Foundation; (b) regulation of cell proliferation and tissue formation, a nationwide study of determinants and markers of successful ageing in Greek centenarians and studies of histone gene expression and acetylation at the National Center for Scientific Research, Demokritos; (c) work on amyloid precursor protein and Presenilin 1 at the University of Athens; (d) oxidative stress-induced DNA damage and the role of oncogenes in senescence at the University of Ioannina; (e) studies in the connective tissue at the University of Patras; (f) proteomic studies at the Biomedical Sciences Research Center Alexander Fleming; (g) work on Caenorhabditis elegans at the Foundation for Research and Technology; (h) the role of ultraviolet radiation in skin ageing at Andreas Sygros Hospital; (i) follow-up studies in healthy elderly at the Athens Home for the Aged; and (j) socio-cultural aspects of ageing at the National School of Public Health. These research activities are well recognized by the international scientific community as it is evident by the group's very good publication records as well as by their direct funding from both European Union and USA. This article summarizes these research activities and discuss future directions and efforts towards the further development of the ageing field in Greece.

Clusterin/apolipoprotein J is a novel biomarker of cellular senescence that does not affect the proliferative capacity of human diploid fibroblasts.

Normal human fibroblasts have a limited replicative potential in culture and eventually reach a state of irreversible growth arrest, termed senescence. In a previous study aiming to identify genes that are differentially regulated during cellular senescence we have cloned clusterin/apolipoprotein J (Apo J), a 80 kDa secreted glycoprotein. In the current report we pursue our studies and show that senescence of human diploid fibroblasts is accompanied by up-regulation of both Apo J mRNA and protein levels, but with no altered biogenesis, binding partner profile or intracellular distribution of the two Apo J forms detected. To analyze the causal relationship between senescence and Apo J protein accumulation, we stably overexpressed the Apo J gene in primary as well as in SV40 T antigen-immortalized human fibroblasts and we showed no alteration of the proliferative capacity of the transduced cells. Despite previous reports on tumor-derived cell lines, overexpression of Apo J in human fibroblasts did not provide protection against apoptosis or growth arrest induced by hydrogen peroxide. Overall, our results suggest that Apo J overexpression does not induce senescence but it is rather a secondary consequence of the senescence phenotype. To our knowledge this is the first report that provides a functional analysis of human Apo J during replicative senescence.

Differential sorting of constitutively co-secreted proteins in the ovarian follicle cells of Drosophila.

Conventional and freeze-fracture electron microscopy, immuno-electron microscopy of ovarian cryosections and confocal immunofluorescence were used to analyze the ovarian distribution of the major protein classes being secreted by the follicle cells during the vitellogenic and choriogenic stages of Drosophila oogenesis. Our results clearly demonstrated that at vitellogenic stages the follicle cells co-secrete constitutively vitelline membrane and yolk proteins that are either sorted into distinct secretory vesicles or they are segregated in different parts of bipartite vesicles by differential condensation. Following their exocytosis only the vitelline membrane proteins are incorporated into the forming vitelline membrane. The yolk proteins (along with their hemolymph circulating counterparts) diffuse through gaps amongst the incomplete vitelline membrane and are internalized through endocytosis by the oocyte where they are finally stored into modified lysosomes referred to as alpha-yolk granules. The unexpected immunolocalization of vitelline membrane antigens in the associated body of the alpha-yolk granules may indicate that this structure is a transient repository for the proteins being internalized into the oocyte along with the yolk proteins. In the early choriogenic follicle cells the vitelline membrane and early chorion proteins were found to be co-secreted and to be evenly intermixed into the same secretory vesicles. These findings illuminate new details concerning the follicle cells secretory and oocyte endocytic pathways and provide for the first time evidence for condensation-mediated sorting of constitutively secreted proteins in Drosophila.

Biochemical and immunocytochemical analysis of vitellogenesis in the olive fruit fly Dacus (bactrocera) oleae (Diptera: Tephritidae).

Synthesis and selective accumulation of the major yolk proteins in the developing oocytes of the species Dacus oleae (Diptera: Tephritidae) was studied biochemically and by immunoelectron microscopy. In the hemolymph of adult females, two yolk proteins precursors (or vitellogenins) have been detected. They each exhibit a similar molecular weight and isoelectric point to their respective mature yolk proteins (or vitellins), while electrophoretic analysis of their synthetic profile shows that their levels in the hemolymph increase rapidly during development. Immunogold electron microscopy of ovarian sections, revealed that the hemolymph vitellogenins reach the oocyte through enlarged inter-follicular spaces and demonstrated vitellogenin synthesis by the follicle cells of the vitellogenic follicles. The newly synthesized vitellogenins follow a distinct secretory pathway into these cells as compared to other components being synthesized at the same time (e.g. the vitelline envelope proteins), since they were found in secretory vesicles that appeared to be differentiated from those destined to participate in the vitelline envelope. The vitellogenin-containing vesicles exocytose their contents directionally into the follicle cell/vitelline envelope boundary, and subsequently the vitellogenins diffuse among the gaps of the forming vitelline envelope and reach the oocyte plasma membrane. Their internalization by the oocyte includes the formation of an endocytic complex consisting of coated pits, coated vesicles, endosomes, transitional yolk bodies, and finally mature yolk bodies, in which the storage of the vitellins and other yolk proteins occur. These results are discussed in relation to data obtained from other Dipteran species.

Immunolocalization of the temporally "early" secreted major structural chorion proteins, Dvs38 and Dvs36, in the eggshell layers and regions of Drosophila virilis.

We have shown by means of conventional electron microscopy that the eggshell of Drosophila virilis at the main body of the laid egg consists of the vitelline membrane and the multilayered chorion, which includes the wax layer, the innermost chorionic layer, the endochorion, and the exochorion, while several specialized regions of the eggshell are seen across the anterior-posterior axis of the egg. Biochemical analysis revealed the existence of six quantitatively enriched chorion proteins. Among them, Dvs38 and Dvs36 are synthesized when the innermost chorionic layer and the endochorion are assembled. Immunogold electron microscopy has shown that these two proteins are incorporated in the morphologically complete vitelline membrane apparently through an intercalation process and represent structural components of the endochorion in all the specialized regions of the eggshell. Additionally, by cytochemical means, the enzyme eggshell peroxidase, which is synthesized in parallel with Dvs38 and Dvs36, has been identified as a structural component of the innermost chorionic layer and the endochorion. These findings suggest a complex protein-protein recognition pattern during the formation of the eggshell since the cosecretion of its components (i.e., Dvs38, Dvs36 chorion proteins and eggshell peroxidase) does not recommend their colocalization in the eggshell sublayers and the timing of their synthesis is not related to their final position on the eggshell (i.e., the identification of Dvs38 and Dvs36 chorion proteins as vitelline membrane components).

The formation of the functional chorion structure of Drosophila virilis involves inercalation of the "middle" and "late" major chorion proteins: a general model for chorion assembly in Drosophilidae.

We have shown by in vitro development of staged follicles in the presence of tritiated proline, as well as by immunoblot analysis, that the Dvs19 and DVS15 chorion proteins of Drosophila virilis are synthesized by the follicular epithelium and are incorporated in the chorion at the terminal stages of choriogenesis. Light microscopy immunolocalization has revealed that these two proteins represent structural components of the endochorion in all the specialized regions of the eggshell, while, contrary to their temporal pattern of secretion, electron microscopic immunolocalization has shown that these two proteins participate in endochorionic structures formed before the onset of their synthesis, thus suggesting an intercalation process during chorion formation. Furthermore, double immunolocalizations demonstrated that cosecreted proteins do not coexist in all secretory vesicles of the follicle cells, indicating that probably the final association of these chorionic components occurs extracellularly. On the basis of these data and those presented in the accompanying paper [Trougakos, I.P., and Margaritis, L.H. (1998) Immunolocalization of the temporally "early" secreted major structural chorion proteins, Dvs38 and Dvs36, in the eggshell layers and regions of Drosophila virilis, J. Struct. Biol. 123, 000-000.], a model for chorion assembly in Drosophilidae is proposed.

Changing patterns of bacterial nosocomial infections: a nine-year study in a general hospital.

Surveillance data on 12,944 bacterial isolates derived from nosocomial infections, reported to the Department of Microbiology and Infectious Diseases of the Hellenic Air Force and VA General Hospital over a 9-year period (1986-1994), were analyzed by the use of a microbial infection control software system. Overall, the isolation rate of Escherichia coli decreased from 25.2% in 1986 to 18.2% in 1994 and Proteus spp. from 5.3 to 2.6%. Remarkably, Pseudomonas spp. increased from 7.2 to 11.3%, Enterobacter spp. from 1.6 to 5.1%, Klebsiella spp. from 5.9 to 7.8% and Enterococcus spp. from 3 to 7.4%. Interestingly, the above phenomenon was paralleled by a significant increase in resistance rate to various antibiotics. Specifically, Staphylococcus aureus and coagulase-negative staphylococci, though they did not display any significant variation in isolation rates, showed an alarming increase in resistance rate to oxacillin, from 11 and 21% in 1986 to 51 and 75% in 1994, respectively. Enterococcus spp. sensitivity to vancomycin remained unlatered at 90%. The above-mentioned serious shift towards more resistant bacteria should be a matter of consideration.