Carol E. Cass

The equilibrative nucleoside transporter family, SLC29

The human SLC29 family of proteins contains four members, designated equilibrative nucleoside transporters (ENTs) because of the properties of the first-characterised family member, hENT1. They belong to the widely-distributed eukaryotic ENT family of equilibrative and concentrative nucleoside/nucleobase transporters and are distantly related to a lysosomal membrane protein, CLN3, mutations in which cause neuronal ceroid lipofuscinosis. A predicted topology of 11 transmembrane helices with a cytoplasmic N-terminus and an extracellular C-terminus has been experimentally confirmed for hENT1. The best-characterised members of the family, hENT1 and hENT2, possess similar broad substrate specificities for purine and pyrimidine nucleosides, but hENT2 in addition efficiently transports nucleobases. The ENT3 and ENT4 isoforms have more recently also been shown to be genuine nucleoside transporters. All four isoforms are widely distributed in mammalian tissues, although their relative abundance varies: ENT2 is particularly abundant in skeletal muscle. In polarised cells ENT1 and ENT2 are found in the basolateral membrane and, in tandem with concentrative transporters of the SLC28 family, may play a role in transepithelial nucleoside transport. The transporters play key roles in nucleoside and nucleobase uptake for salvage pathways of nucleotide synthesis, and are also responsible for the cellular uptake of nucleoside analogues used in the treatment of cancers and viral diseases. In addition, by regulating the concentration of adenosine available to cell surface receptors, they influence many physiological processes ranging from cardiovascular activity to neurotransmission.

Gastric Adenocarcinoma: Review and Considerations for Future Directions

Objective:This update reviews the epidemiology and surgical management, and the controversies of gastric adenocarcinoma. We provide the relevance of outcome data to surgical decision-making and discuss the application of gene-expression analysis to clinical practice. Summary Background Data:Gastric cancer mortality rates have remained relatively unchanged over the past 30 years, and gastric cancer continues to be one of the leading causes of cancer-related death. Well-conducted studies have stimulated changes to surgical decision-making and technique. Microarray studies linked to predictive outcome models are poised to advance our understanding of the biologic behavior of gastric cancer and improve surgical management and outcome. Methods:We performed a review of the English gastric adenocarcinoma medical literature (1980–2003). This review included epidemiology, pathology and staging, surgical management, issues and controversies in management, prognostic variables, and the application of outcome models to gastric cancer. The results of DNA microarray analysis in various cancers and its predictive abilities in gastric cancer are considered. Results:Prognostic studies have provided valuable data to better the understanding of gastric cancer. These studies have contributed to improved surgical technique, more accurate pathologic characterization, and the identification of clinically useful prognostic markers. The application of microarray analysis linked to predictive models will provide a molecular understanding of the biology driving gastric cancer. Conclusions:Predictive models generate important information allowing a logical evolution in the surgical and pathologic understanding and therapy for gastric cancer. However, a greater understanding of the molecular changes associated with gastric cancer is needed to guide surgical and medical therapy.

NUCLEOSIDE TRANSPORTERS : MOLECULAR BIOLOGY AND IMPLICATIONS FOR THERAPEUTIC DEVELOPMENT

The uptake of nucleosides (or nucleobases) is essential for nucleic acid synthesis in many human cell types and in parasitic organisms that cannot synthesize nucleotides de novo. The transporters responsible are also the route of entry for many cytotoxic nucleoside analogues used in cancer and viral chemotherapy. Moreover, by regulating adenosine concentrations in the vicinity of its cell-surface receptors, nucleoside transporters profoundly affect neurotransmission, vascular tone and other processes. The recent molecular characterization of two families of human nucleoside transporters has provided new insights into the mechanisms of natural nucleoside and drug uptake and into future developments of improved therapies.

Human Equilibrative Nucleoside Transporter 1 Levels Predict Response to Gemcitabine in Patients With Pancreatic Cancer

BACKGROUND & AIMS The human equilibrative nucleoside transporter (hENT1) protein transports gemcitabine into cells. Small retrospective studies in pancreatic cancer suggest that levels of hENT1 protein or messenger RNA may have prognostic value. We studied the predictive value of hENT1 levels in a cohort of pancreatic adenocarcinoma patients from the large prospective randomized adjuvant treatment trial RTOG9704. METHODS In RTOG9704, 538 patients were assigned randomly, after surgical resection, to groups that were given either gemcitabine or 5-fluorouracil (5-FU). Immunohistochemistry for hENT1 was performed on a tissue microarray of 229 resected pancreatic tumors from RTOG9704 and scored as having no staining, low staining, or high staining. Associations between hENT1 protein and treatment outcome were analyzed by unconditional logistic regression analysis using the chi-square test and the Cox proportional hazards model. RESULTS HENT1 expression was associated with overall and disease-free survival in a univariate (hazard ratio [HR], 0.51; 95% confidence interval [CI], 0.29-0.91; P= .02; and HR, 0.57; 95% CI, 0.32-1.00; P= .05) and multivariate model in the group given gemcitabine (HR, 0.40; 95% CI, 0.22-0.75; P= .004; and HR, 0.39; 95% CI, 0.21-0.73; P= .003). hENT1 expression was not associated with survival in the group given 5-FU. CONCLUSIONS In this prospective randomized trial, hENT1 protein expression was associated with increased overall survival and disease-free survival in pancreatic cancer patients who received gemcitabine, but not in those who received 5-FU. These findings are supported by preclinical data; the gemcitabine transporter hENT1 is therefore a molecular and mechanistically relevant predictive marker of benefit from gemcitabine in patients with resected pancreatic cancer.

The Absence of Human Equilibrative Nucleoside Transporter 1 Is Associated with Reduced Survival in Patients With Gemcitabine- Treated Pancreas Adenocarcinoma

Purpose: Gemcitabine monotherapy is the standard palliative chemotherapy for pancreatic adenocarcinoma. Gemcitabine requires plasma membrane nucleoside transporter proteins to efficiently enter cells and exert it cytotoxicity. In vitro studies have demonstrated that deficiency of human equilibrative nucleoside transporter 1 (hENT1), the most widely abundant and distributed nucleoside transporter in human cells, confers resistance to gemcitabine toxicity, but the distribution and abundance of nucleoside transporters in normal and malignant pancreatic tissue is unknown. Experimental Design: We studied tumor blocks from normal pancreas and 21 Alberta patients with gemcitabine-treated pancreatic cancer. Immunohistochemistry on the formalin-fixed, paraffin-embedded tissues was performed with specific hENT1 and human Concentrative Nucleoside Transporter 3 monoclonal antibodies and scored by a pathologist blinded to clinical outcomes. Results: hENT1 was detected in normal Langerhan cells and lymphocytes but not in normal glandular elements. Patients in whom all adenocarcinoma cells had detectable hENT1 had significantly longer median survivals from gemcitabine initiation than those for whom hENT1 was absent in a proportion (10 to 100%) of adenocarcinoma cells (median survival, 13 versus 4 months, P = 0.01). Immunohistochemistry for human Concentrative Nucleoside Transporter 3 revealed moderate to high-intensity staining in all adenocarcinoma tissue samples. Conclusions: Patients with pancreatic adenocarcinoma with uniformly detectable hENT1 immunostaining have a significantly longer survival after gemcitabine chemotherapy than tumors without detectable hENT1. Immunohistochemistry for hENT1 shows promise as a molecular predictive assay to appropriately select patients for palliative gemcitabine chemotherapy but requires formal validation in prospective, randomized trials.

Functional Characterization of Novel Human and Mouse Equilibrative Nucleoside Transporters (hENT3 and mENT3) Located in Intracellular Membranes

The first mammalian examples of the equilibrative nucleoside transporter family to be characterized, hENT1 and hENT2, were passive transporters located predominantly in the plasma membranes of human cells. We now report the functional characterization of members of a third subgroup of the family, from human and mouse, which differ profoundly in their properties from previously characterized mammalian nucleoside transporters. The 475-residue human and mouse proteins, designated hENT3 and mENT3, respectively, are 73% identical in amino acid sequence and possess long N-terminal hydrophilic domains that bear typical (DE)XXXL(LI) endosomal/lysosomal targeting motifs. ENT3 transcripts and proteins are widely distributed in human and rodent tissues, with a particular abundance in placenta. However, in contrast to ENT1 and ENT2, the endogenous and green fluorescent protein-tagged forms of the full-length hENT3 protein were found to be predominantly intracellular proteins that co-localized, in part, with lysosomal markers in cultured human cells. Truncation of the hydrophilic N-terminal region or mutation of its dileucine motif to alanine caused the protein to be relocated to the cell surface both in human cells and in Xenopus oocytes, allowing characterization of its transport activity in the latter. The protein proved to be a broad selectivity, low affinity nucleoside transporter that could also transport adenine. Transport activity was relatively insensitive to the classical nucleoside transport inhibitors nitrobenzylthioinosine, dipyridamole, and dilazep and was sodium ion-independent. However, it was strongly dependent upon pH, and the optimum pH value of 5.5 probably reflected the location of the transporter in acidic, intracellular compartments.

Nucleoside anticancer drugs: the role of nucleoside transporters in resistance to cancer chemotherapy.

The clinical efficacy of anticancer nucleoside drugs depends on a complex interplay of transporters mediating entry of nucleoside drugs into cells, efflux mechanisms that remove drugs from intracellular compartments and cellular metabolism to active metabolites. Nucleoside transporters (NTs) are important determinants for salvage of preformed nucleosides and mediated uptake of antimetabolite nucleoside drugs into target cells. The focus of this review is the two families of human nucleoside transporters (hENTs, hCNTs) and their role in transport of cytotoxic chemotherapeutic nucleoside drugs. Resistance to anticancer nucleoside drugs is a major clinical problem in which NTs have been implicated. Single nucleotide polymorphisms (SNPs) in drug transporters may contribute to interindividual variation in response to nucleoside drugs. In this review, we give an overview of the functional and molecular characteristics of human NTs and their potential role in resistance to nucleoside drugs and discuss the potential use of genetic polymorphism analyses for NTs to address drug resistance.

MOLECULAR CLONING AND FUNCTIONAL CHARACTERIZATION OF NITROBENZYLTHIOINOSINE (NBMPR)-SENSITIVE (ES) AND NBMPR-INSENSITIVE (EI) EQUILIBRATIVE NUCLEOSIDE TRANSPORTER PROTEINS (RENT1 AND RENT2) FROM RAT TISSUES

Equilibrative nucleoside transport processes in mammalian cells are either nitrobenzylthioinosine (NBMPR)-sensitive (es) or NBMPR-insensitive (ei). Previously, we isolated a cDNA from human placenta encoding the 456-residue glycoprotein hENT1. When expressed inXenopus oocytes, hENT1 mediated es-type transport activity and was inhibited by coronary vasoactive drugs (dipyridamole and dilazep) that may compete with nucleosides and NBMPR for binding to the substrate binding site. We now report the molecular cloning and functional expression of es and eihomologs of hENT1 from rat tissues; rENT1 (457 residues) was 78% identical to hENT1 in amino acid sequence, and rENT2 (456 residues) was 49–50% identical to rENT1/hENT1 and corresponded to a full-length form of the delayed-early proliferative response gene product HNP36, a protein of unknown function previously cloned in truncated form. rENT1 was inhibited by NBMPR (IC50 = 4.6 nm at 10 μm uridine), whereas rENT2 was NBMPR-insensitive (IC50 > 1 μm). Both proteins mediated saturable uridine influx (K m = 0.15 and 0.30 mm, respectively), were broadly selective for purine and pyrimidine nucleosides, including adenosine, and were relatively insensitive to inhibition by dipyridamole and dilazep (IC50> 1 μm). These observations demonstrate thates and ei nucleoside transport activities are mediated by separate, but homologous, proteins and establish a function for the HNP36 gene product.

The ENT family of eukaryote nucleoside and nucleobase transporters: recent advances in the investigation of structure/function relationships and the identification of novel isoforms

The first examples of the equilibrative nucleoside transporter (ENT) family were characterized in human tissues at the molecular level only 4 years ago. Since that time, the identification of homologous proteins by functional cloning and genome analysis has revealed that the family is widely distributed in eukaryotes. Family members are predicted to possess 11 transmembrane helices (TMs), and recent investigations on the mammalian ENTs have implicated the TM 3-6 region in solute recognition. Whilst the name of the family reflects the properties ofits prototypical member hENT1, an equilibrative transporter of nucleosides, some family members can also transport nucleobases and some are proton-dependent, concentrative transporters. In addition to their role in nucleoside salvage, ENTs are targets for coronary vasodilator drugs and act as routes for uptake of cytotoxic drugs in humans and protozoa. This paper summarizes current knowledge of the family and reports on the identification of a novel mammalian ENT isoform, designated ENT3, from mouse and human tissues.

Recent advances in the molecular biology of nucleoside transporters of mammalian cells

Nucleosides are hydrophilic molecules and require specialized transport proteins for permeation of cell membranes. There are two types of nucleoside transport processes: equilibrative bidirectional processes driven by chemical gradients and inwardly directed concentrative processes driven by the sodium electrochemical gradient. The equilibrative nucleoside transport processes (es, ei) are found in most mammalian cell types, whereas the concentrative nucleoside transport processes (cit, cif, cib, csg, cs) are present primarily in specialized epithelia. Using a variety of cloning strategies and functional expression in oocytes of Xenopus laevis, we have isolated and characterized cDNAs encoding the rat and human nucleoside transporter proteins of the four major nucleoside transport processes of mammalian cells (es, ei, cit, cif). From the sequence relationships of these proteins with each other and with sequences in the public data bases, we have concluded that the equilibrative and concentrative nucleoside transport processes are mediated by members of two previously unrecognized groups of integral membrane proteins, which we have designated the equilibrative nucleoside transporter (ENT) and the concentrative nucleoside transporter (CNT) protein families. This review summarizes the current state of knowledge in the molecular biology of the ENT and CNT protein families, focusing on the characteristics of the four human (h) and rat (r) nucleoside transport proteins (r/hENT1, r/hENT2, r/hCNT1, r/hCNT2).