Up-regulation of Skp2 after prostate cancer cell adhesion to basement membranes results in BRCA2 degradation and cell proliferation

Aberrant interaction of carcinoma cells with basement membranes (BM) is a fundamental pathophysiological process that initiates a series of events resulting in cancer cell invasion and metastasis. In this report, we describe the results of our investigations pertaining to the events triggered by the adhesion of normal (PNT1A) and highly metastatic (PC-3) prostate cells onto BM proteins. Unlike PNT1A, PC-3 cells adhered avidly to Matrigel BM matrix as well as to isolated collagen type IV, laminin, and heparan sulfate proteoglycan perlecan, main BM components. This aberrantly increased cancer cell adhesion resulted in sustained BRCA2 protein depletion and vigorous cell proliferation, a cascade triggered by beta1 integrin-mediated phosphatidylinositol 3-kinase activation leading to BRCA2 degradation in the proteasome. This latter effect was orchestrated by phosphatidylinositol 3-kinase-dependent up-regulation of Skp2, a subunit of the Skp1-Cul1-F-box protein ubiquitin complex that directly associates with BRCA2 as demonstrated by coimmunoprecipitation assays, determines its ubiquitination, and ultimately targets it for proteasomal degradation. Inhibition of Skp2 expression by small interference RNA prevented BRCA2 depletion and inhibited the trophic effect upon cell proliferation. These results provide additional evidence on the role of BRCA2 as a modulator of cancer cell growth and elucidate the molecular mechanisms involved in its down-regulation in cancer cells when interacting with BM, a crucial step in the biology of metastasis. Furthering the understanding of this molecular pathway may prove valuable in designing new therapeutic strategies aimed at modifying the natural history of prostate carcinoma.     

PED/PEA-15 controls fibroblast motility and wound closure by ERK1/2-dependent mechanisms

Cell migration is dependent on the control of signaling events that play significant roles in creating contractile force and in contributing to wound closure. We evaluated wound closure in fibroblasts from mice overexpressing (TgPED) or lacking ped/pea-15 (KO), a gene overexpressed in patients with type 2 diabetes. Cultured skin fibroblasts isolated from TgPED mice showed a significant reduction in the ability to recolonize wounded area during scratch assay, compared to control fibroblasts. This difference was observed both in the absence and in the presence of mytomicin C, an inhibitor of mitosis. In time-lapse experiments, TgPED fibroblasts displayed about twofold lower velocity and diffusion coefficient, as compared to controls. These changes were accompanied by reduced spreading and decreased formation of stress fibers and focal adhesion plaques. At the molecular level, TgPED fibroblasts displayed decreased RhoA activation and increased abundance of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2). Inhibition of ERK1/2 activity by PD98059 restored RhoA activation, cytoskeleton organization and cell motility, and almost completely rescued wound closure of TgPED fibroblasts. Interestingly, skin fibroblasts isolated from KO mice displayed an increased wound closure ability. In vivo, healing of dorsal wounds was delayed in TgPED and accelerated in KO mice. Thus, PED/PEA-15 may affect fibroblast motility by a mechanism, at least in part, mediated by ERK1/2.     

Adiponectin oligomerization state and adiponectin receptors airway expression in chronic obstructive pulmonary disease

Adiponectin (Acrp30) shows several beneficial properties and circulates as different oligomers. The role of Acrp30 in lung is not fully clear, but a link with chronic obstructive pulmonary disease (COPD) has been highlighted. In this study, we analyzed the anthropometrical and biochemical features and evaluated total Acrp30 levels of a COPD cohort without metabolic complications compared to healthy controls. In addition, being the oligomerization state critical for its biological activities, we characterized the pattern of Acrp30 circulating oligomers focusing on the high molecular weight (HMW) oligomers to verify whether it correlates to COPD. Finally, we investigated AdipoR1 and AdipoR2 expression in lung from COPD. Interestingly, we found for the first time that the oligomerization state of Acrp30 is altered in COPD; particularly, we observed that the higher levels of Acrp30 are associated with a significant and specific increase of HMW. In addition, we demonstrated the presence of AdipoRs with a lower expression of AdipoR2 compared to AdipoR1. In conclusion, we demonstrated that in COPD, the higher levels of Acrp30 are associated with the significantly increase of HMW representing the most biologically active forms. The important role of Acrp30 in pathophysiological conditions of lung is supported also by the modulation of AdipoRs with the down regulation of AdipoR2. The low expression of AdipoR2 could suggest a specific role of this receptor, mainly implicated in Acrp30 effects on inflammation and oxidative stress. Thus, total Acrp30, HMW and its receptors could be considered critical targets to improve diagnostic and therapeutic strategies for lung diseases.     

Catalase T and Cu,Zn-superoxide dismutase in the acetic acid-induced programmed cell death in Saccharomyces cerevisiae

To investigate the role of catalase and superoxide dismutase (SOD) in the acetic acid (AA) induced yeast programmed cell death (AA-PCD), we compared Saccharomyces cerevisiae cells (C-Y) and cells individually over-expressing catalase T (CTT1-Y) and Cu,Zn-SOD (SOD1-Y) with respect to cell survival, hydrogen peroxide (H2O2) levels and enzyme activity as measured up to 200 min after AA treatment. AA-PCD does not occur in CTT1-Y, where H2O2 levels were lower than in C-Y and the over-expressed catalase activity decreased with time. In SOD1-Y, AA-PCD was exacerbated; high H2O2 levels were found, SOD activity increased early, remaining constant en route to AA-PCD, but catalase activity was strongly reduced.     

Pyrosequencing Unveils Cystic Fibrosis Lung Microbiome Differences Associated with a Severe Lung Function Decline

Chronic airway infection is a hallmark feature of cystic fibrosis (CF) disease. In the present study, sputum samples from CF patients were collected and characterized by 16S rRNA gene-targeted approach, to assess how lung microbiota composition changes following a severe decline in lung function. In particular, we compared the airway microbiota of two groups of patients with CF, i.e. patients with a substantial decline in their lung function (SD) and patients with a stable lung function (S). The two groups showed a different bacterial composition, with SD patients reporting a more heterogeneous community than the S ones. Pseudomonas was the dominant genus in both S and SD patients followed by Staphylococcus and Prevotella. Other than the classical CF pathogens and the most commonly identified non-classical genera in CF, we found the presence of the unusual anaerobic genus Sneathia. Moreover, the oligotyping analysis revealed the presence of other minor genera described in CF, highlighting the polymicrobial nature of CF infection. Finally, the analysis of correlation and anti-correlation networks showed the presence of antagonism and ecological independence between members of Pseudomonas genus and the rest of CF airways microbiota, with S patients showing a more interconnected community in S patients than in SD ones. This population structure suggests a higher resilience of S microbiota with respect to SD, which in turn may hinder the potential adverse impact of aggressive pathogens (e.g. Pseudomonas). In conclusion, our findings shed a new light on CF airway microbiota ecology, improving current knowledge about its composition and polymicrobial interactions in patients with CF.     

Organization of Patient Management and Fungal Epidemiology in Cystic Fibrosis

The achievement of a better life for cystic fibrosis (CF) patients is mainly caused by a better management and infection control over the last three decades. Herein, we want to summarize the cornerstones for an effective management of CF patients and to give an overview of the knowledge about the fungal epidemiology in this clinical context in Europe. Data from a retrospective analysis encompassing 66,616 samples from 3235 CF patients followed-up in 9 CF centers from different European countries are shown.     

In the early phase of programmed cell death in Tobacco Bright Yellow 2 cells the mitochondrial adenine nucleotide translocator, adenylate kinase and nucleoside diphosphate kinase are impaired in a reactive oxygen species-dependent manner

To investigate whether and how mitochondria can change in plant programmed cell death (PCD), we used the non-photosynthetic Tobacco Bright Yellow 2 (TBY-2) cells. These can be synchronized to high levels, stand out in terms of growth rate and homogeneity and undergo PCD as a result of heat shock. Using these cells we investigated the activity of certain mitochondrial proteins that have a role in providing ATP and/or other nucleoside triphosphates (NTPs). We show that, already after 2 h from the heat shock, when cell viability remains unaffected, the rate of ADP/ATP exchange due to adenine nucleotide translocator (ANT) activity, and the rate of the reactions catalysed by adenylate kinase (ADK; EC 2.7.4.3) and nucleoside diphosphate kinase (NDPK; EC 2.7.4.6) are inhibited in a non-competitive-like manner. In all cases, externally added ascorbate partially prevented the inhibition. These effects occurred in spite of minor (for ANT) or no changes in the mitochondrial protein levels as immunologically investigated. Interestingly, a decrease of both the steady state level of the ascorbate pool and of the activity of l-galactono-gamma-lactone dehydrogenase (GLDH) (EC 1.3.2.3), the mitochondrial enzyme catalysing the last step of ascorbate biosynthesis, were also found.     

Glutamate Neurotoxicity in Rat Cerebellar Granule Cells Involves Cytochrome c Release from Mitochondria and Mitochondrial Shuttle Impairment

To gain some insight into the mechanism by which glutamate neurotoxicity takes place in cerebellar granule cells, two steps of glucose oxidation were investigated: the electron flow via respiratory chain from certain substrates to oxygen and the transfer of extramitochondrial reducing equivalents via the mitochondrial shuttles. However, cytochrome c release from intact mitochondria was found to occur in glutamate-treated cells as detected photometrically in the supernatant of the cell homogenate suspension. As a result of cytochrome c release, an increase of the oxidation of externally added NADH was found, probably occurring via the NADH-b5 oxidoreductase of the outer mitochondrial membrane. When the two mitochondrial shuttles glycerol 3-phosphate/dihydroxyacetone phosphate and malate/oxaloacetate, devoted to oxidizing externally added NADH, were reconstructed, both were found to be impaired under glutamate neurotoxicity. Consistent early activation in two NADH oxidizing mechanisms, i.e., lactate production and plasma membrane NADH oxidoreductase activity, was found in glutamate-treated cells. In spite of this, the increase in the cell NADH fluorescence was found to be time-dependent, an index of the progressive damage of the cell.     

Mammalian Rif1 contributes to replication stress survival and homology-directed repair

Rif1, originally recognized for its role at telomeres in budding yeast, has been implicated in a wide variety of cellular processes in mammals, including pluripotency of stem cells, response to double-strand breaks, and breast cancer development. As the molecular function of Rif1 is not known, we examined the consequences of Rif1 deficiency in mouse cells. Rif1 deficiency leads to failure in embryonic development, and conditional deletion of Rif1 from mouse embryo fibroblasts affects S-phase progression, rendering cells hypersensitive to replication poisons. Rif1 deficiency does not alter the activation of the DNA replication checkpoint but rather affects the execution of repair. RNA interference to human Rif1 decreases the efficiency of homology-directed repair (HDR), and Rif1 deficiency results in aberrant aggregates of the HDR factor Rad51. Consistent with a role in S-phase progression, Rif1 accumulates at stalled replication forks, preferentially around pericentromeric heterochromatin. Collectively, these findings reveal a function for Rif1 in the repair of stalled forks by facilitating HDR.