Increased ENT2 expression and its association with altered purine metabolism in cell lines derived from different stages of colorectal cancer.

Colorectal cancer (CRC) is one of the most prevalent malignant cancer types worldwide. Although the purine metabolism pathway is vital for cancer cell survival, little is known about the role of equilibrative nucleoside transporter 2 (ENT2) in CRC development and its association with purine metabolites. The aim of the present study was to evaluate the levels of hypoxanthine phosphoribosyl transferase (HPRT), hypoxanthine and uric acid (UA), as well as xanthine oxidase (XO) activity, and investigate their association with ENT2 expression levels in a normal human colon cell line and CRC cell lines derived from different stages of CRC. These analyses were performed using the normal colon CCD-841CoN cell line and a panel of human CRC cell lines comprising SW480, HCT15 and HCT116, which represent Dukes' B, C and D stages, respectively. Reverse transcription-quantitative PCR was performed to determine the level of ENT2 mRNA expression. In cells of all CRC stages, the levels of HPRT and hypoxanthine were significantly higher (P<0.05), while XO activity and UA levels were significantly decreased (P<0.05), compared with those in the CCD-841CoN cell line. ENT2 expression was found to be elevated in cells derived from all stages of CRC. The Dukes' D stage cell line had higher levels of HPRT and hypoxanthine, although its ENT2 level was not significantly lower than that of the Dukes' B and C stage cell lines. Increased levels of HPRT and hypoxanthine in various stages of CRC may indicate an increase in the activity of the salvage pathway. The increased expression of ENT2 implies the importance of the ENT2 protein in facilitating hypoxanthine transport, which is required for enhanced DNA synthesis via hypoxanthine recycling. In conclusion, ENT2 may have potential as a target in the development of CRC therapeutics.

Ent2 Governs Morphogenesis and Virulence in Part through Regulation of the Cdc42 Signaling Cascade in the Fungal Pathogen Candida albicans.

The ability to transition between yeast and filamentous growth states is critical for virulence of the leading human fungal pathogen Candida albicans. Large-scale genetic screens have identified hundreds of genes required for this morphological switch, but the mechanisms by which many of these genes orchestrate this developmental transition remain largely elusive. In this study, we characterized the role of Ent2 in governing morphogenesis in C. albicans. We showed that Ent2 is required for filamentous growth under a wide range of inducing conditions and is also required for virulence in a mouse model of systemic candidiasis. We found that the epsin N-terminal homology (ENTH) domain of Ent2 enables morphogenesis and virulence and does so via a physical interaction with the Cdc42 GTPase-activating protein (GAP) Rga2 and regulation of its localization. Further analyses revealed that overexpression of the Cdc42 effector protein Cla4 can overcome the requirement for the ENTH-Rga2 physical interaction, indicating that Ent2 functions, at least in part, to enable proper activation of the Cdc42-Cla4 signaling pathway in the presence of a filament-inducing cue. Overall, this work characterizes the mechanism by which Ent2 regulates hyphal morphogenesis in C. albicans, unveils the importance of this factor in enabling virulence in an in vivo model of systemic candidiasis and adds to the growing understanding of the genetic control of a key virulence trait. IMPORTANCE Candida albicans is a leading human fungal pathogen that can cause life-threatening infections in immunocompromised individuals, with mortality rates of ~40%. The ability of this organism to grow in both yeast and filamentous forms is critical for the establishment of systemic infection. Genomic screens have identified many genes required for this morphological transition, yet our understanding of the mechanisms that regulate this key virulence trait remains incomplete. In this study, we characterized Ent2 as a core regulator of C. albicans morphogenesis. We show that Ent2 regulates hyphal morphogenesis through an interaction between its ENTH domain and the Cdc42 GAP, Rga2, which signals through the Cdc42-Cla4 signaling pathway. Finally, we show that the Ent2 protein, and specifically its ENTH domain, is required for virulence in a mouse model of systemic candidiasis. Overall, this work identifies Ent2 as a key regulator of filamentation and virulence in C. albicans.

The Hypoxia-Adenosine Link during Myocardial Ischemia-Reperfusion Injury.

Despite increasing availability and more successful interventional approaches to restore coronary reperfusion, myocardial ischemia-reperfusion injury is a substantial cause of morbidity and mortality worldwide. During myocardial ischemia, the myocardium becomes profoundly hypoxic, thus causing stabilization of hypoxia-inducible transcription factors (HIF). Stabilization of HIF leads to a transcriptional program that promotes adaptation to hypoxia and cellular survival. Transcriptional consequences of HIF stabilization include increases in extracellular production and signaling effects of adenosine. Extracellular adenosine functions as a signaling molecule via the activation of adenosine receptors. Several studies implicated adenosine signaling in cardioprotection, particularly through the activation of the Adora2a and Adora2b receptors. Adenosine receptor activation can lead to metabolic adaptation to enhance ischemia tolerance or dampen myocardial reperfusion injury via signaling events on immune cells. Many studies highlight that clinical strategies to target the hypoxia-adenosine link could be considered for clinical trials. This could be achieved by using pharmacologic HIF activators or by directly enhancing extracellular adenosine production or signaling as a therapy for patients with acute myocardial infarction, or undergoing cardiac surgery.

Molecular Basis of Nucleobase Transport Systems in Mammals.

Nucleobases are water-soluble compounds that need specific transporters to cross biological membranes. Cumulative evidence based on studies using animal tissues and cells indicates that the carrier-mediated transport systems for purine and pyrimidine nucleobases can be classified into the following two types: concentrative transport systems that mediate nucleobase transport depending on the sodium ion concentration gradient; and other systems that mediate facilitated diffusion depending on the concentration gradient of the substrate. Recently, several molecular transporters that are involved in both transport systems have been identified. The function and activity of these transporters could be of pharmacological significance considering the roles that they play not only in nucleotide synthesis and metabolism but also in the pharmacokinetics and delivery of a variety of nucleobase analogues used in anticancer and antiviral drug therapy. The present review provides an overview of the recent advances in our understanding of the molecular basis of nucleobase transport systems, focusing on the transporters that mediate purine nucleobases, and discusses the involvement of intracellular metabolism in purine nucleobase transport and chemotherapy using ganciclovir.

Novel nuclear hENT2 isoforms regulate cell cycle progression via controlling nucleoside transport and nuclear reservoir.

Nucleosides participate in many cellular processes and are the fundamental building blocks of nucleic acids. Nucleoside transporters translocate nucleosides across plasma membranes although the mechanism by which nucleos(t)ides are translocated into the nucleus during DNA replication is unknown. Here, we identify two novel functional splice variants of equilibrative nucleoside transporter 2 (ENT2), which are present at the nuclear envelope. Under proliferative conditions, these splice variants are up-regulated and recruit wild-type ENT2 to the nuclear envelope to translocate nucleosides into the nucleus for incorporation into DNA during replication. Reduced presence of hENT2 splice variants resulted in a dramatic decrease in cell proliferation and dysregulation of cell cycle due to a lower incorporation of nucleotides into DNA. Our findings support a novel model of nucleoside compartmentalisation at the nuclear envelope and translocation into the nucleus through hENT2 and its variants, which are essential for effective DNA synthesis and cell proliferation.

The role of the adenosinergic system in lung fibrosis.

Adenosine (ADO) is a retaliatory metabolite that is expressed in conditions of injury or stress. During these conditions ATP is released at the extracellular level and is metabolized to adenosine. For this reason, adenosine is defined as a "danger signal" for cells and organs, in addition to its important role as homeostatic regulator. Its physiological functions are mediated through interaction with four specific transmembrane receptors called ADORA1, ADORA2A, ADORA2B and ADORA3. In the lungs of mice and humans all four adenosine receptors are expressed with different roles, having pro- and anti-inflammatory roles, determining bronchoconstriction and regulating lung inflammation and airway remodeling. Adenosine receptors can also promote differentiation of lung fibroblasts into myofibroblasts, typical of the fibrotic event. This last function suggests a potential involvement of adenosine in the fibrotic lung disease processes, which are characterized by different degrees of inflammation and fibrosis. Idiopathic pulmonary fibrosis (IPF) is the pathology with the highest degree of fibrosis and is of unknown etiology and burdened by lack of effective treatments in humans.