Role of Adenosine Kinase in the Biological (Antiviral and Anticellular) Activities of Adenosine Analogues

Abstract

A paired adenosine kinase-positive/adenosine kinase-negative cell system is proposed to distinguish those adenosine analogues that need to be phosphorylated to exert their biological effects from those that are mainly targeted at S-adenosyl-L-homocysteine hydrolase.     

Disaccharide Nucleosides And Their Enzymatic And Chemical Incorporation Into Oligonucleotides

Abstract

A high yield synthesis of different O-ribofuranosylnucleosides has been achieved. Kinetics of the acid-catalysed hydrolysis of disaccharide nucleosides has been studied. Chemical and enzymatic incorporation of 2′-O-ribofuranosyl-nucleoside residue into oligonucleotides was investigated.     

Proteomic Profiling of Triple-negative Breast Carcinomas in Combination With a Three-tier Orthogonal Technology Approach Identifies Mage-A4 as Potential Therapeutic Target in Estrogen Receptor Negative Breast Cancer

Breast cancer is a very heterogeneous disease, encompassing several intrinsic subtypes with various morphological and molecular features, natural history and response to therapy. Currently, molecular targeted therapies are available for estrogen receptor (ER)⁻ and human epidermal growth factor receptor 2 (Her2)-positive breast tumors. However, a significant proportion of primary breast cancers are negative for ER, progesterone receptor (PgR), and Her2, comprising the triple negative breast cancer (TNBC) group. Women with TNBC have a poor prognosis because of the aggressive nature of these tumors and current lack of suitable targeted therapies. As a consequence, the identification of novel relevant protein targets for this group of patients is of great importance. Using a systematic two dimensional (2D) gel-based proteomic profiling strategy, applied to the analysis of fresh TNBC tissue biopsies, in combination with a three-tier orthogonal technology (two dimensional PAGE/silver staining coupled with MS, two dimensional Western blotting, and immunohistochemistry) approach, we aimed to identify targetable protein markers that were present in a significant fraction of samples and that could define therapy-amenable sub-groups of TNBCs. We present here our results, including a large cumulative database of proteins based on the analysis of 78 TNBCs, and the identification and validation of one specific protein, Mage-A4, which was expressed in a significant fraction of TNBC and Her2-positive/ER negative lesions. The high level expression of Mage-A4 in the tumors studied allowed the detection of the protein in the tumor interstitial fluids as well as in sera. The existence of immunotherapeutics approaches specifically targeting this protein, or Mage-A protein family members, and the fact that we were able to detect its presence in serum suggest novel management options for TNBC and human epidermal growth factor receptor 2 positive/estrogen receptor negative patients bearing Mage-A4 positive tumors.Breast cancer, although a very heterogeneous disease, can be divided into three therapeutically relevant fundamental disease entities, simply based on estrogen receptor (ER) and human epidermal growth factor receptor 2 (Her2)¹ status (i.e. ER⁺ and/or Her2⁺, and ER⁻Her2⁻), as the major currently available breast cancer therapeutic options are based on the ability to target these proteins. Hormone receptor positive and hormone receptor negative breast cancers are disease entities with distinct morphological, genetic and biological behavior (1). Hormone receptor negative tumors, which constitute ∼30% of primary breast cancers, tend to be high-grade, more frequently BRCA1 and TP53 mutated, and, more importantly, are not amenable to endocrine therapy. Her2 is amplified in ∼18–20% of breast cancers, and is more frequently observed in hormone receptor negative tumors. Her2 amplification is associated with worse prognosis (higher rate of recurrence and mortality) in patients with newly diagnosed breast cancer who do not receive any adjuvant systemic therapy. Her2 status is also predictive for several systemic therapies, particularly for agents that target Her2. The development of a humanized monoclonal antibody against Her2 (trastuzumab) has resulted in reduction of the risk of recurrence and mortality in patients with Her2 amplification (2, 3). Although trastuzumab is considered one of the most effective targeted therapies currently available in oncology, a significant number of patients with Her2-overexpressing breast cancer do not benefit from it (4, 5).Breast tumors that do not express ER, PgR, or Her2 (ER⁻ PgR⁻ Her2⁻), as determined by immunohistochemistry (IHC), are generally referred to as triple negative breast cancers (TNBCs), and they are not candidates for targeted therapies (endocrine therapy or trastuzumab). Although TNBCs account for a relatively small proportion of breast cancer cases (10–15%), they are responsible for a disproportionate number of breast cancer deaths. TNBC tumors form a recognizable prognostic group of breast cancer with aggressive behavior that currently lacks the benefit of available systemic therapy (6–8). Given the need to develop molecular criteria to reproducibly categorize molecular breast tumor subtypes at the protein level and the lack of targeted therapies available to treat patients bearing TNBCs, we have implemented a systematic proteomics approach to identify, characterize, and evaluate proteins present in triple-negative tumors that could constitute an appropriate therapeutic target for the clinical management of this group of patients. To this end, based on the analysis of 78 individual TNBC samples, we have established a large, cumulative, 2D-PAGE database of proteins expressed by TNBCs, including some that could be of potential therapeutic value. Comparison of this TNBC protein database with protein databases of other breast cancer subtypes previously established by our laboratory allowed us to single out a number of proteins preferentially expressed in TNBCs for which targeted therapeutics exist. In this report we further focused on the characterization of one such target, the cancer/testis antigen, melanoma-associated antigen 4 - Mage-A4.Cancer/testis antigens (CTAs) are expressed in a large variety of tumor types, whereas their expression in normal tissues is restricted to male germ cells, which are immune-privileged because of their lack of or low expression of human leukocyte antigen (HLA) molecules (9). Several studies have shown the existence of natural cellular and humoral responses against some CTAs, indicating that they are appropriate targets for vaccine-based cancer immunotherapy (10–12). So far, the use of CTAs in immunotherapeutic approaches to cancer treatment has been tested in more than 60 early phase clinical trials, with varying success, and a few candidate products have reached late-stage clinical trials. One such candidate vaccine, Astuprotimut-R (GSK-249553), a Mage-A3 antigen-specific cancer immunotherapeutic agent, is currently under clinical evaluation by GlaxoSmithKline in the largest-ever treatment trial in lung cancer, called MAGRIT (Mage-A3 as Adjuvant nonsmall cell lunG canceR ImmunoTherapy) (13).At present, CTAs comprise about 150 members, more than half of which are encoded by large, recently expanded families on chromosome X (14; see also CTDatabase at www.cta.lncc.br; last accessed 01.09.2012). These genes are organized into clusters and have undergone rapid evolution, possibly because of positive selection. The biological functions of CTAs are not fully understood, but emerging evidence suggest that they direct the proliferation, differentiation, and survival of human germ line cells and may have similar effect in cancer cells. Mage-A4 protein belongs to the Mage-A family of CT antigens. The Mage-A family is composed by 12 proteins (14, 15) and many members of the Mage-A family of CTAs have been associated with cancer, including breast cancer (14, 16, 17). However, past studies reported mostly on MAGE genes rather than protein expression, or on the expression of Mage protein families and not on any given specific protein.In this paper we describe the identification of Mage-A4 in breast tumor biopsies using 2D PAGE coupled with MS proteomics, and follow the protein localization from the tumor cells, to the tumor microenvironment, and to the serum of a patient. Using a three-tier orthogonal technology approach that combined 2D PAGE silver staining coupled with MS, with 2D Western blotting, and IHC, we showed that high level Mage-A4 expression in breast tumors occurs almost exclusively in the receptor negative disease (TNBC and Her2⁺ER⁻PgR⁻). The existence of immunotherapeutic approaches targeting MAGE protein family members (Mage-A4 specific or with broader specificity) and the fact that we were able to detect its presence in serum suggest novel management options for patients bearing Mage-A4 positive TNBCs and Her2⁺ER⁻PgR⁻ tumors.     

The evolving systemic and local biomarker milieu at different stages of disease progression in rat collagen-induced arthritis

Rats with collagen-induced arthritis (CIA) were necropsied on 14 occasions from 4 days after induction to 27 days after disease onset to evaluate the kinetics of local (joint protein extracts) and systemic (serum) levels of inflammatory and pro-erosive factors. Systemic increases in α1 acid glycoprotein and KC/GRO together with systemic and local enrichment of interleukin (IL)-1β, IL-6, CCL2, transforming growth factor (TGF)-β and elevated IL-1α and IL-18 in joint extracts preceded the onset of clinical disease. Systemic upregulation of IL-1β, IL-6, TGF-β CCL2, RANKL and prostaglandin E₂ (PGE₂) during acute and/or chronic CIA coincided with systemic leukocytosis and a CD4+ T-cell increase in blood and spleen. In contrast, progression of joint erosions during clinical CIA was associated with intra-articular increases in IL-1α/β, IL-6, IL-18, CCL2, KC/GRO and RANKL, and a dramatic decline in osteoprotegerin (OPG). These data indicate that systemic and local events in inflammatory arthritis can be discrete processes, driven by multiple cellular and humoral mediators with distinct temporospatial profiles.     

Alien parasite hitchhikes to Patagonia on invasive bumblebee

The worldwide trade in bumblebees can lead to the spread of diseases, which in turn has been claimed as a factor in bumblebee decline. Populations of the introduced Bombus terrestris, which invaded NW Patagonia, Argentina, in 2006, harbor the highly pathogenic protozoan Apicystis bombi. We asked whether A. bombi had been co-introduced with B. terrestris, and if so, whether spillover occurred to the two resident bumblebee species in the region: the introduced European Bombus ruderatus and the native Bombus dahlbomii. We searched for A. bombi by means of PCR in samples of B. ruderatus and B. dahlbomii collected before and after the invasion of B. terrestris and in samples of the latter. We found no A. bombi in samples of B. ruderatus and B. dahlbomii collected before B. terrestris invasion, whereas post invasion, A. bombi was present in all 3 species. The identity of the parasite was established by sequencing the 18S region, which was identical for the three bumblebee species and also matched the European sequence, confirming it to be A. bombi. This is the first report of A. bombi in B. ruderatus and B. dahlbomii. Moreover, our results suggest that Patagonia had been free of A. bombi until this parasite was co-introduced with B. terrestris, and spilled over in situ to these two previously resident species. Finally, our findings provide indirect circumstantial evidence of a potential link between the population collapse and geographic retraction of B. dahlbomii and the introduction of this novel parasite.     

Herbivore-mediated facilitation alters composition and increases richness and diversity in ruderal communities

Little is known about positive interactions among members of herbaceous plant communities initiating secondary succession (i.e., ruderal communities). Here, we explored the possibility that Euphorbia schickendantzii (Euphorbia), a latex-containing herb, facilitates other ruderals by protecting them from herbivores in recently plowed and overgrazed sites in central Argentina. To test this hypothesis, we compared plant number, height, reproductive output, and herbivore damage for four species when associated with Euphorbia versus in adjacent open zones without Euphorbia. Additionally, we classified species in the community according to their palatability, and compared community composition, richness, and diversity between Euphorbia and open zones. Dominant (66 % relative abundance) and highly palatable species exhibited increased plant number, size, and fecundity, and decreased herbivory when associated with Euphorbia relative to non-Euphorbia zones. In contrast, a physically and chemically well-defended species showed greater number of individuals in the open and no differences in herbivory between sampling zones. In detrended correspondence analysis, ordination scores of most palatable species were closer to Euphorbia, while those of most unpalatable species were closer to the open. Community composition differed between areas, with six species (25 % of the community) occurring exclusively with Euphorbia and three other species occurring only in open zones. Additionally, richness and diversity were greater in communities associated with Euphorbia than in those associated with non-Euphorbia zones. These results support our hypothesis, highlight the importance of facilitation in altering community-level responses, and indicate that positive interactions can play a more significant role in organizing terrestrial ruderal communities than previously recognized.     

Elimination of Aspergillus fumigatus conidia from the airways of mice with allergic airway inflammation

Background

Aspergillus fumigatus conidia can exacerbate asthma symptoms. Phagocytosis of conidia is a principal component of the host antifungal defense. We investigated whether allergic airway inflammation (AAI) affects the ability of phagocytic cells in the airways to internalize the resting fungal spores.

Methods

Using BALB/c mice with experimentally induced AAI, we tested the ability of neutrophils, macrophages, and dendritic cells to internalize A. fumigatus conidia at various anatomical locations. We used light microscopy and differential cell and conidium counts to determine the ingestion potential of neutrophils and macrophages present in bronchoalveolar lavage (BAL). To identify phagocyte-conidia interactions in conducting airways, conidia labeled with tetramethylrhodamine-(5-(and-6))-isothiocyanate were administered to the oropharyngeal cavity of mice. Confocal microscopy was used to quantify the ingestion potential of Ly-6G⁺ neutrophils and MHC II⁺ antigen-presenting cells located in the intraepithelial and subepithelial areas of conducting airways.

Results

Allergen challenge induced transient neutrophil recruitment to the airways. Application of A. fumigatus conidia at the acute phase of AAI provoked recurrent neutrophil infiltration, and consequently increased the number and the ingestion potential of the airway neutrophils. In the absence of recurrent allergen or conidia provocation, both the ingestion potential and the number of BAL neutrophils decreased. As a result, conidia were primarily internalized by alveolar macrophages in both AAI and control mice at 24 hours post-inhalation. Transient influx of neutrophils to conducting airways shortly after conidial application was observed in mice with AAI. In addition, the ingestion potential of conducting airway neutrophils in mice with induced asthma exceeded that of control mice. Although the number of neutrophils subsequently decreased, the ingestion capacity remained elevated in AAI mice, even at 24 hours post-conidia application.

Conclusions

Aspiration of allergen to sensitized mice enhanced the ingestion potential of conducting airway neutrophils. Such activation primes neutrophils so that they are sufficient to control dissemination of non-germinating A. fumigatus conidia. At the same time, it can be a reason for the development of sensitivity to fungi and subsequent asthma exacerbation.     

Integrating frugivory and animal movement: a review of the evidence and implications for scaling seed dispersal

General principles about the consequences of seed dispersal by animals for the structure and dynamics of plant populations and communities remain elusive. This is in part because seed deposition patterns emerge from interactions between frugivore behaviour and the distribution of food resources, both of which can vary over space and time. Here we advocate a frugivore‐centred, process‐based, synthetic approach to seed dispersal research that integrates seed dispersal ecology and animal movement across multiple spatio‐temporal scales. To guide this synthesis, we survey existing literature using paradigms from seed dispersal and animal movement. Specifically, studies are discussed with respect to five criteria: selection of focal organisms (animal or plant); measurement of animal movement; characterization of seed shadow; animal, plant and environmental factors included in the study; and scales of the study. Most studies focused on either frugivores or plants and characterized seed shadows directly by combining gut retention time with animal movement data or indirectly by conducting maternity analysis of seeds. Although organismal traits and environmental factors were often measured, they were seldom used to characterize seed shadows. Multi‐scale analyses were rare, with seed shadows mostly characterized at fine spatial scales, over single fruiting seasons, and for individual dispersers. Novel animal‐ and seed‐tracking technologies, remote environmental monitoring tools, and advances in analytical methods can enable effective implementation of a hierarchical mechanistic approach to the study of seed dispersal. This kind of mechanistic approach will provide novel insights regarding the complex interplay between the factors that modulate animal behaviour and subsequently influence seed dispersal patterns across spatial and temporal scales.     

Cigarette smoke metabolically promotes cancer,via autophagy and premature aging in the host stromal microenvironment

Cigarette smoke has been directly implicated in the disease pathogenesis of a plethora of different human cancer subtypes, including breast cancers. The prevailing view is that cigarette smoke acts as a mutagen and DNA damaging agent in normal epithelial cells, driving tumor initiation. However, its potential negative metabolic effects on the normal stromal microenvironment have been largely ignored. Here, we propose a new mechanism by which carcinogen-rich cigarette smoke may promote cancer growth, by metabolically “fertilizing” the host microenvironment. More specifically, we show that cigarette smoke exposure is indeed sufficient to drive the onset of the cancer-associated fibroblast phenotype via the induction of DNA damage, autophagy and mitophagy in the tumor stroma. In turn, cigarette smoke exposure induces premature aging and mitochondrial dysfunction in stromal fibroblasts, leading to the secretion of high-energy mitochondrial fuels, such as L-lactate and ketone bodies. Hence, cigarette smoke induces catabolism in the local microenvironment, directly fueling oxidative mitochondrial metabolism (OXPHOS) in neighboring epithelial cancer cells, actively promoting anabolic tumor growth. Remarkably, these autophagic-senescent fibroblasts increased breast cancer tumor growth in vivo by up to 4-fold. Importantly, we show that cigarette smoke-induced metabolic reprogramming of the fibroblastic stroma occurs independently of tumor neo-angiogenesis. We discuss the possible implications of our current findings for the prevention of aging-associated human diseases and, especially, common epithelial cancers, as we show that cigarette smoke can systemically accelerate aging in the host microenvironment. Finally, our current findings are consistent with the idea that cigarette smoke induces the “reverse Warburg effect,” thereby fueling “two-compartment tumor metabolism” and oxidative mitochondrial metabolism in epithelial cancer cells.