Nucleoside Transporter Proteins Research Articles

A novel start-loss mutation of the SLC29A3 gene in a consanguineous family with H syndrome: clinical characteristics, in silico analysis and literature review

BackgroundThe SLC29A3 gene, which encodes a nucleoside transporter protein, is primarily located in intracellular membranes. The mutations in this gene can give rise to various clinical manifestations, including H syndrome, dysosteosclerosis, Faisalabad histiocytosis, and pigmented hypertrichosis with insulin-dependent diabetes. The aim of this study is to present two Iranian patients with H syndrome and to describe a novel start-loss mutation in SLC29A3 gene.MethodsIn this study, we employed whole-exome sequencing (WES) as a method to identify genetic variations that contribute to the development of H syndrome in a 16-year-old girl and her 8-year-old brother. These siblings were part of an Iranian family with consanguineous parents. To confirmed the pathogenicity of the identified variant, we utilized in-silico tools and cross-referenced various databases to confirm its novelty. Additionally, we conducted a co-segregation study and verified the presence of the variant in the parents of the affected patients through Sanger sequencing.ResultsIn our study, we identified a novel start-loss mutation (c.2T > A, p.Met1Lys) in the SLC29A3 gene, which was found in both of two patients. Co-segregation analysis using Sanger sequencing confirmed that this variant was inherited from the parents. To evaluate the potential pathogenicity and novelty of this mutation, we consulted various databases. Additionally, we employed bioinformatics tools to predict the three-dimensional structure of the mutant SLC29A3 protein. These analyses were conducted with the aim of providing valuable insights into the functional implications of the identified mutation on the structure and function of the SLC29A3 protein.ConclusionOur study contributes to the expanding body of evidence supporting the association between mutations in the SLC29A3 gene and H syndrome. The molecular analysis of diseases related to SLC29A3 is crucial in understanding the range of variability and raising awareness of H syndrome, with the ultimate goal of facilitating early diagnosis and appropriate treatment. The discovery of this novel biallelic variant in the probands further underscores the significance of utilizing genetic testing approaches, such as WES, as dependable diagnostic tools for individuals with this particular condition.

Genetic disruption of nucleoside transporter 4 reveals its critical roles in malaria parasite sporozoite functions

ABSTRACT All protozoan parasites are lacking the pathway to synthesize purines de novo and therefore they depend on their host cells to provide purines. A number of highly conserved nucleoside transporter (NT) proteins are encoded in malaria parasite genomes, of which NT1 is characterized in Plasmodium falciparum and P. yoelii as a plasma membrane protein that is responsible for salvage of purines from the host, and NT2 is an endoplasmic membrane NT protein. Whereas NT3 is only present in primate malaria parasites, little is known about NT4, which is conserved in all malaria parasite species. Herein, we targeted NT4 gene for deletion in P. berghei. NT4 knockout parasites developed normally as blood stages, ookinetes and formed oocysts with sporozoites compared with wild-type (WT) P. berghei ANKA parasites. However, nt4(-) sporozoites showed significantly decreased egress from oocysts to hemolymph, significant reduction of colonization of the salivary glands, and complete abolishment of infection of the mammalian host by salivary gland and hemolymph sporozoites. Therefore, we identify NT4 as a NT that is important, not for replication and growth, but for sporozoite infectivity functions.

TH2.6 Evaulation of the prognostic utility for hENT 1 transmembranous protein in resected hilar cholangiocarcinoma patients

Abstract Aims Human equilibriative nucleoside transporter protein 1 (hENT1) facilitates nucleoside transport into the cell. Immunohistochemically-detected hENT1 is increased in cholangiocarcinoma tumour cells and in highly metabolising cells. The Mackey 10D7G2 hybridoma has demonstrated prognostic utility in Pancreatic Ductal Adenocarcinoma. The Proteintech Polyclonal hENT1 antibody's prognostic utility has not been assessed. Cellular Ki67 expression is linked to tumour proliferation. This study aims to assess the antibodies’ prognostic utility for resectable hilar cholangiocarcinoma. Methods Between 2009–2016, 54 patients underwent resection for peri-hilar cholangiocarcinoma. Formalin-Fixed Paraffin Embedded blocks from 44 specimens were retrieved. A Tissue-Matched Array was constructed and stained for each antibody. H-scores were utilised to determine expression intensity. Correlation of expression was determined by Pearson correlation co-efficient and Chi-squared. Silencing RNA for transfected HepG2 cell-lines was used to determine accurate hENT1 staining by the Proteintech antibody. Demographic and survival characteristics were acquired from a prospectively held database, and were calculated with global log-rank calculations. Results There was significant correlation between the Mackey and Proteintech antibodies (p<0.001), and between the Proteintech antibody and Ki67 expression (p= 0.02). Knockdown of hENT1 with silencing RNA transfected HepG2 cells was confirmed by Western blot. The antibodies (Mackey; Proteintech; Ki67) showed no significance for predicting OS (p= 0.75; 0.63; 0.22 respectively). Nodal stage (p= 0.03) and tumour differentiation (p= 0.02) showed prognostic utility. Conclusion While the Proteintech antibody demonstrates concordance with the 10D7G2 antibody in determining hENT1 expression, they didn't demonstrate significant prognostic ability. Standard histopathological co-variates retain prognostic utility within the cohort.

Inborn Errors of Nucleoside Transporter (NT)-Encoding Genes (SLC28 and SLC29).

The proper regulation of nucleotide pools is essential for all types of cellular functions and depends on de novo nucleotide biosynthesis, salvage, and degradation pathways. Despite the apparent essentiality of these processes, a significant number of rare diseases associated with mutations in genes encoding various enzymes of these pathways have been already identified, and others are likely yet to come. However, knowledge on genetic alterations impacting on nucleoside and nucleobase transporters is still limited. At this moment three gene-encoding nucleoside and nucleobase transporter proteins have been reported to be mutated in humans, SLC29A1, SLC29A3, and SLC28A1, impacting on the expression and function of ENT1, ENT3, and CNT1, respectively. ENT1 alterations determine Augustine-null blood type and cause ectopic calcification during aging. ENT3 deficiency translates into various clinical manifestations and syndromes, altogether listed in the OMIM catalog as histiocytosis-lymphoadenopathy plus syndrome (OMIM#602782). CNT1 deficiency causes uridine-cytidineuria (URCTU) (OMIM#618477), a unique type of pyrimidineuria with an as yet not well-known clinical impact. Increasing knowledge on the physiological, molecular and structural features of these transporter proteins is helping us to better understand the biological basis behind the biochemical and clinical manifestations caused by these deficiencies. Moreover, they also support the view that some metabolic compensation might occur in these disturbances, because they do not seem to significantly impact nucleotide homeostasis, but rather other biological events associated with particular subtypes of transporter proteins.

Effect of Transporter Genotype on the Rate of Caffeine Absorption

Caffeine is the most widely used psychoactive drug in the world, with the majority of the world’s population consuming it on a daily basis. Individuals respond very differently to caffeine, with both environmental factors and genetic variation of proteins involved in metabolism, mode of action, and absorption believed to modulate caffeine’s effects. For example, a single nucleotide polymorphism (rs762551) of the gene for the liver enzyme CYP1A2 has been reported to influence caffeine outcomes, with “slow” metabolizers having reduced benefits of caffeine during exercise and increased risk of heart attack. Our working hypothesis is that single nucleotide polymorphisms in the nucleoside transporter protein ENT1, encoded by SLC29A1, may play a role in caffeine absorption, a topic that has widespread medical significance. In this study, human subjects were genotyped at the SLC29A1 locus for SNP rs760370, previously suggested to modulate the efficacy of nucleoside analogue therapy. Subjects were also phenotyped via ELISA to determine how fast they actually absorb caffeine. We have observed significant variability in tmax (time to maximum concentration) among our subjects and are assessing a potential relationship between tmaxand a subject’s genotype to determine whether some genotypes correlate with faster rates of caffeine absorption. Overall, this work may contribute to a better understanding of the variable effects of caffeine consumption, as well as inform the timing of caffeine dosing for those seeking to enhance alertness and/or performance.

Localization and functional characterization of SLC28 transporter variants

Nucleoside metabolites and synthetic nucleoside analogues used in the treatment of cancer and viral diseases cross lipid bilayers via specialized nucleoside transport proteins that are either equilibrative or concentrative. The concentrative nucleoside transporter family (CNT, SLC28) consists of three subtypes, CNT1, CNT2, and CNT3 (SLC28A1, SLC28A2, and SLC28A3, respectively), that transport nucleosides together with an inwardly directed sodium gradient. Using the crystal structure of concentrative nucleoside transporter from Vibrio cholerae as a reference (Johnson et al, Nature 2012), models for human SLC28 and naturally occurring variants were generated.

O-L09 Determining the Prognostic Utility of Immuno-histochemically detected Trans-membranous Human Equilibriative Nucleoside Transporter 1 (hENT1) in Patients Undergoing Hilar Cholangiocarcinoma Resection

Abstract Background Human equilibriative nucleoside transporter protein 1 (hENT1) is a trans-membranous protein which facilitates nucleoside transport in to the cell. Immunohistochemically-detected hENT1 abundance is increased in cholangiocarcinoma tumour cells compared to matched non-tumour cells and increased in highly metabolising cells. The privately-held Mackey 10D7G2 hybridoma has demonstrated prognostic utility in Pancreatic Ductal Adenocarcinoma patients. The commercially available Proteintech Polyclonal hENT1 antibody’s prognostic utility has not been previously assessed. Cellular Ki67 expression has been linked to mitotic indices of tumour proliferation. This proof-of-concept study aims to assess the antibodies prognostic utility for hilar cholangiocarcinoma patients undergoing surgical resection. Methods Between February 2009 and February 2016 54 patients underwent resection for peri-hilar cholangiocarcinoma. Formalin-Fixed Paraffin Embedded (FFPE) blocks from a sub-set of 44 resected specimens were retrieved. Appropriate areas of tumour were sampled from the blocks and a Tissue-Matched Array (TMA) was constructed. The TMA underwent staining for each antibody. H-scores were utilised to determine intensity of expression. Correlation of expression between antibodies was determined by Pearson correlation co-efficient and Chi-squared where appropriate. Silencing RNA transfected HepG2 cell-lines was used to determine hENT1 staining by the Proteintech antibody. Demographic and survival characteristics for the patients were acquired from a prospectively held database linked to Hospital Episode Statistics. Survival characteristics were calculated with global log-rank calculations. Results There was significant correlation between the Mackey 10D7G2 and the Proteintech antibodies (p < 0.001). There was significant correlation between the Proteintech hENT1 antibody expression and Ki67 expression (p = 0.02). Knockdown of hENT1 with silencing RNA transfected HepG2 cells was confirmed by Western blot in a time-dependent fashion over 72 hours. The antibodies (Mackey; Proteintech; Ki67) did not achieve significance for predicting OS (p = 0.75; 0.63; 0.22 respectively). Nodal stage (p = 0.03) and grade of tumour differentiation (p = 0.02) were the univariate tumour variables with prognostic utility. Conclusions While the Proteintech antibody demonstrates concordance with the 10D7G2 antibody in determining hENT1 expression the antibodies did not demonstrate significant prognostic ability in this proof-of-concept study. Standard histopathological co-variates retain prognostic utility within the cohort.

A genome-scale CRISPR/Cas9 knockout screening reveals SH3D21 as a sensitizer for gemcitabine

Gemcitabine, 2′,2′-difluoro-2′-deoxycytidine, is used as a pro-drug in treatment of variety of solid tumour cancers including pancreatic cancer. After intake, gemcitabine is transferred to the cells by the membrane nucleoside transporter proteins. Once inside the cells, it is converted to gemcitabine triphosphate followed by incorporation into DNA chains where it causes inhibition of DNA replication and thereby cell cycle arrest and apoptosis. Currently gemcitabine is the standard drug for treatment of pancreatic cancer and despite its widespread use its effect is moderate. In this study, we performed a genome-scale CRISPR/Cas9 knockout screening on pancreatic cancer cell line Panc1 to explore the genes that are important for gemcitabine efficacy. We found SH3D21 as a novel gemcitabine sensitizer implying it may act as a therapeutic target for improvement of gemcitabine efficacy in treatment of pancreatic cancer.

A novel homozygous frame-shift mutation in the SLC29A3 gene: a new case report and review of literature

BackgroundThe SLC29A3 gene, encoding a nucleoside transporter protein, is found in intracellular membranes. Based on the literatures, mutations in this gene cause a wide range of clinical manifestations including H syndrome, pigmented hypertrichosis with insulin dependent diabetes, Faisalabad histiocytosis, and dysosteosclerosis. However, all these disorders with their different names and terminologies are actually the same entity termed H syndrome.Case presentationWe report four GJB2 and GJB6 negative deaf patients from two Iranian related families who present the associated symptoms of SLC29A3-disorder. Whole Exome Sequencing (WES) using Next Generation Illumina Sequencing was used to enrich all exons of protein-coding genes as well as some other important genomic regions in one of studied patients. A novel homozygous frame-shift mutation c.307-308delTT (p.Phe103fs) in exon 3 of SLC29A3 gene was identified in a 35 years old man with profound hearing loss, camptodactyly, rheumatoid arthritis and delayed puberty without any skin changes, short stature and insulin dependent diabetes mellitus. The mutation found was also confirmed by Sanger sequencing in other studied patients and their healthy parents. In compared to proband, however the clinical manifestations of these patients were different, indicating variable expressivity of mutant SLC29A3 gene as well as possible involvement of other modifier genes.ConclusionThe present study uncovered a rare novel homozygous frame-shift mutation c.307-308delTT in SLC29A3 gene of four related patients with various manifestation of SLC29A3-disorder. Such studies can help to conduct genetic counseling and subsequently, prenatal diagnosis more accurately for individuals at the high risk of these types of genetic disorders.

Adjuvant gemcitabine versus 5-fluorouracil/folinic acid based on hENT1 immunostaining in curative resected pancreatic adenocarcinoma: A biomaker stratified trial.

308 Background: The human equilibrating nucleoside transporter (hENT1) protein transports gemcitabine into the cell. However, role of hENT1 as a predictive biomarker for gemcitabine responsiveness in pancreatic adenocarcinoma (PDA) is still needed to be elucidated, although several studies had been conducted. We investigated whether hENT1 has a predictable role on adjuvant chemotherapy responsiveness in PDA according to stratification of hENT1 immunostaining. Methods: The 44 patients who underwent curative surgery (R0 or R1) due to PDA at Seoul National University Bundang Hospital from May 2015 to June 2017 were enrolled prospectively. The hENT1 expressions were measured by immunochemical staining (SP120-rabbit monoclonal antibody, Ventana). According to hENT1 immunostaing (≥ 50% vs. < 50%), higher hENT1 group received gemcitabine [1,000mg/m2 (day 1, 8, 15) q 4 weeks x 6 cycles] and lower hENT1 group received [5-FU 425mg/m2 (day 1-5) plus folinic acid 20 mg/m2 (day 1) q 4 weeks x 6 cycles]. Primary outcome was overall survival (OS) and secondary outcomes were toxicity and recurrence free survival (RFS). Results: Among 44 patients, 5 patients were excluded due to withdrew consent (3 patients) and toxicities (2 patients in 5FU/LV group). Eighteen patients in the higher hENT1 group and twenty-six patients in the lower hENT1 group were followed up for 24.1 months. Our data showed OS was 97.4% at 12 months and 86.4% at 24 months. Moreover, RFS was 76.8% at 12 months and 64.2% at 24 months. Although it is preliminary data, it showed better OS and RFS compared to previous study (Neoptolemos JP et al., JAMA 2010) (OS, 61.3% at 12 months, 30.7% at 24 months; RFS, 58.7% at 12 months and 30.1% at 24 months). Conclusions: Adjuvant chemotherapy based on hENT1 immunostaining stratification provides excellent survival in resected PDA. Therefore, hENT1 immunostaining should be used for making a decision for adjuvant chemotherapy remimen in PDA. Clinical trial information: NCT02486497.

Metabolite systems profiling identifies exploitable weaknesses in retinoblastoma.

Retinoblastoma (RB) is a childhood eye cancer. Currently, chemotherapy, local therapy, and enucleation are the main ways in which these tumors are managed. The present work is the first study that uses constraint-based reconstruction and analysis approaches to identify and explain RB-specific survival strategies, which are RB tumor specific. Importantly, our model-specific secretion profile is also found in RB1-depleted human retinal cells invitro and suggests that novel biomarkers involved in lipid metabolism may be important. Finally, RB-specific synthetic lethals have been predicted as lipid and nucleoside transport proteins that can aid in novel drug target development.

Metabolic engineering of Escherichia coli for high-yield uridine production

Uridine is a kind of pyrimidine nucleoside that has been widely applied in the pharmaceutical industry. Although microbial fermentation is a promising method for industrial production of uridine, an efficient microbial cell factory is still lacking. In this study, we constructed a metabolically engineered Escherichia coli capable of high-yield uridine production. First, we developed a CRISPR/Cas9-mediated chromosomal integration strategy to integrate large DNA into the E. coli chromosome, and a 9.7 kb DNA fragment including eight genes in the pyrimidine operon of Bacillus subtilis F126 was integrated into the yghX locus of E. coli W3110. The resultant strain produced 3.3 g/L uridine and 4.5 g/L uracil in shake flask culture for 32 h. Subsequently, five genes involved in uridine catabolism were knocked out, and the uridine titer increased to 7.8 g/L. As carbamyl phosphate, aspartate, and 5′-phosphoribosyl pyrophosphate are important precursors for uridine synthesis, we further modified several metabolism-related genes and synergistically improved the supply of these precursors, leading to a 76.9% increase in uridine production. Finally, nupC and nupG encoding nucleoside transport proteins were deleted, and the extracellular uridine accumulation increased to 14.5 g/L. After 64 h of fed-batch fermentation, the final engineered strain UR6 produced 70.3 g/L uridine with a yield and productivity of 0.259 g/g glucose and 1.1 g/L/h, respectively. To the best of our knowledge, this is the highest uridine titer and productivity ever reported for the fermentative production of uridine.

Intestinal Nucleoside Transporters: Function, Expression, and Regulation.

The gastrointestinal tract is the absorptive organ for nutrients found in foods after digestion. Nucleosides and, to a lesser extent nucleobases, are the late products of nucleoprotein digestion. These metabolites are absorbed by nucleoside (and nucleobase) transporter (NT) proteins. NTs are differentially distributed along the gastrointestinal tract showing also polarized expression in epithelial cells. Concentrative nucleoside transporters (CNTs) are mainly located at the apical side of enterocytes, whereas equilibrative nucleoside transporters (ENTs) facilitate the basolateral efflux of nucleosides and nucleobases to the bloodstream. Moreover, selected nucleotides and the bioactive nucleoside adenosine act directly on intestinal cells modulating purinergic signaling. NT-polarized insertion is tightly regulated. However, not much is known about the modulation of intestinal NT function in humans, probably due to the lack of appropriate cell models retaining CNT functional expression. Thus, the possibility of nutritional regulation of intestinal NTs has been addressed using animal models. Besides the nutrition-related role of NT proteins, orally administered drugs also need to cross the intestinal barrier, this event being a major determinant of drug bioavailability. In this regard, NT proteins might also play a role in pharmacology, thereby allowing the absorption of nucleoside- and nucleobase-derived drugs. The relative broad selectivity of these membrane transporters also suggests clinically relevant drug-drug interactions when using combined therapies. This review focuses on all these physiological and pharmacological aspects of NT protein biology. © 2017 American Physiological Society. Compr Physiol 8:1003-1017, 2018.

Who Is Who in Adenosine Transport.

Extracellular adenosine concentrations are regulated by a panel of membrane transporters which, in most cases, mediate its uptake into cells. Adenosine transporters belong to two gene families encoding Equilibrative and Concentrative Nucleoside Transporter proteins (ENTs and CNTs, respectively). The lack of appropriate pharmacological tools targeting every transporter subtype has introduced some bias on the current knowledge of the role of these transporters in modulating adenosine levels. In this regard, ENT1, for which pharmacology is relatively well-developed, has often been identified as a major player in purinergic signaling. Nevertheless, other transporters such as CNT2 and CNT3 can also contribute to purinergic modulation based on their high affinity for adenosine and concentrative capacity. Moreover, both transporter proteins have also been shown to be under purinergic regulation via P1 receptors in different cell types, which further supports its relevance in purinergic signaling. Thus, several transporter proteins regulate extracellular adenosine levels. Moreover, CNT and ENT proteins are differentially expressed in tissues but also in particular cell types. Accordingly, transporter-mediated fine tuning of adenosine levels is cell and tissue specific. Future developments focusing on CNT pharmacology are needed to unveil transporter subtype-specific events.

Adenosine causes read-through into the late region of the HPV16 genome in a guanosine-dependent manner

Adenosine plays an important role in cell death and differentiation as well as in tumorigenesis and the intra- and extra-cellular levels range from nanomolar to millimolar levels under various physiological or pathophysiological conditions. Here we report that adenosine can activate HPV16 late gene expression in a dose- and time-dependent manner, but only in the presence of guanosine. This activation occurred within hours after addition of the nucleosides and was primarily dependent on the ENT1 nucleoside transporter protein. Induction of HPV16 late gene expression was mainly the result of increased read-through at the early HPV16 polyadenylation signal into the late region of the HPV16 genome, thereby producing HPV16 late L2 mRNAs. The effect of guanosine and adenosine on HPV16 late gene expression was mediated by the increased binding to HPV16 mRNAs and nuclear export of the cellular HuR protein. Our results demonstrate that nucleosides can affect HPV16 gene expression.

Substituted cysteine accessibility method (SCAM) analysis of the transport domain of human concentrative nucleoside transporter 3 (hCNT3) and other family members reveals features of structural and functional importance

The human SLC28 family of concentrative nucleoside transporter (CNT) proteins has three members: hCNT1, hCNT2, and hCNT3. Na+-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 transports both pyrimidine and purine nucleosides utilizing Na+ and/or H+ electrochemical gradients. Escherichia coli CNT family member NupC resembles hCNT1 in permeant selectivity but is H+-coupled. Using heterologous expression in Xenopus oocytes and the engineered cysteine-less hCNT3 protein hCNT3(C−), substituted cysteine accessibility method analysis with the membrane-impermeant thiol reactive reagent p-chloromercuribenzene sulfonate was performed on the transport domain (interfacial helix 2, hairpin 1, putative transmembrane domain (TM) 7, and TM8), as well as TM9 of the scaffold domain of the protein. This systematic scan of the entire C-terminal half of hCNT3(C−) together with parallel studies of the transport domain of wild-type hCNT1 and the corresponding TMs of cysteine-less NupC(C−) yielded results that validate the newly developed structural homology model of CNT membrane architecture for human CNTs, revealed extended conformationally mobile regions within transport-domain TMs, identified pore-lining residues of functional importance, and provided evidence of an emerging novel elevator-type mechanism of transporter function.

P3.03-035 Prognostic Role of hENT1 and RRM1 in Patients with Advanced Pleural Mesothelioma Treated with Second Line Gemcitabine Based Regimens: Topic: Mesothelioma Clinical

Nucleoside transporter proteins mediates the transport of nucleosides and nucleoside analog drugs across the plasma membrane. The human equilibrative nucleoside transporter 1 (hENT1) is a nucleoside transporter protein that mediates cellular entry of gemcitabine, cytarabine, and fludarabine. The hENT1 expression has been demonstrated to be related with prognosis and activity of gemcitabine-based therapy in breast, ampullary, lung, and pancreatic cancer. In the same way, RRM1 encodes the regulatory subunit of ribonucleotide reductase and is a molecular target of gemcitabine. Previous studies showed increased RRM1 expression on continuous exposure of cell lines to gemcitabine and suggested improved survival for patients with low RRM1 expression. We measured hENT1 and RRM1 messenger RNA (mRNA) levels by quantitative real-time reverse transcription-polymerase chain reaction in tumor samples from 29 patients with advanced pleural mesothelioma (APM) treated in second line with gemcitabine based chemotherapy correlating these data with clinical parameters and disease outcomes (overall response rate-ORR, progression free survival-PFS and overall survival-OS). The median age was 60-yo (r, 33-70 years), 15 patients were males (51%), 34% of cases undergone debulking surgery and the median follow-up was 13.4 months (95% CI 6.4-34). All patients were treated with Pem based chemotherapy in first line achieving a PFS of 8.1 months (95% CI 3.7-12.6), while PFS with second-line Gem based chemotherapy was 6.3 months (95% CI 3.6-8.8). 62% and 34.5% of patients had low levels of mRNA for hENT1 and RRM1, respectively. On univariate survival analysis, the hENT1 expression was associated with OS (p=0.001) and PFS (p=0.022). Specifically, those patients with overexpression of hENT1 showed a shorter OS p=0.021) and a shorter PFS (p=0.033). In contrast, mRNA expression of RRM1 did not influence the OS (p = 0.44) but it modifies positively the PFS (p=0.034). Multivariate analysis found that combined hENT1 (p=0.001) and RMM1 (p=0.012) predict survival in patients with APM treated with Gem based regimens. hENT1 mRNA expression carries prognostic information in patients with APM and combined with RRM1 holds promise as a predictive biomarkers in gemcitabine treated patients.

The SLC28 (CNT) and SLC29 (ENT) nucleoside transporter families: a 30-year collaborative odyssey.

Specialized nucleoside transporter (NT) proteins are required for passage of nucleosides and hydrophilic nucleoside analogues across biological membranes. Physiologic nucleosides serve as central salvage metabolites in nucleotide biosynthesis, and nucleoside analogues are used as chemotherapeutic agents in the treatment of cancer and antiviral diseases. The nucleoside adenosine modulates numerous cellular events via purino-receptor cell signalling pathways. Human NTs are divided into two structurally unrelated protein families: the SLC28 concentrative nucleoside transporter (CNT) family and the SLC29 equilibrative nucleoside transporter (ENT) family. Human CNTs are inwardly directed Na(+)-dependent nucleoside transporters found predominantly in intestinal and renal epithelial and other specialized cell types. Human ENTs mediate bidirectional fluxes of purine and pyrimidine nucleosides down their concentration gradients and are ubiquitously found in most, possibly all, cell types. Both protein families are evolutionarily old: CNTs are present in both eukaryotes and prokaryotes; ENTs are widely distributed in mammalian, lower vertebrate and other eukaryote species. This mini-review describes a 30-year collaboration with Professor Stephen Baldwin to identify and understand the structures and functions of these physiologically and clinically important transport proteins.

Nucleoside transporter proteins as biomarkers of drug responsiveness and drug targets.

Nucleoside and nucleobase analogs are currently used in the treatment of solid tumors, lymphoproliferative diseases, viral infections such as hepatitis and AIDS, and some inflammatory diseases such as Crohn. Two gene families are implicated in the uptake of nucleosides and nucleoside analogs into cells, SCL28 and SLC29. The former encodes hCNT1, hCNT2, and hCNT3 proteins. They translocate nucleosides in a Na+ coupled manner with high affinity and some substrate selectivity, being hCNT1 and hCNT2 pyrimidine- and purine-preferring, respectively, and hCNT3 a broad selectivity transporter. SLC29 genes encode four members, being hENT1 and hENT2 the only two which are unequivocally implicated in the translocation of nucleosides and nucleobases (the latter mostly via hENT2) at the cell plasma membrane. Some nucleoside-derived drugs can also interact with and be translocated by members of the SLC22 gene family, particularly hOCT and hOAT proteins. Inter-individual differences in transporter function and perhaps, more importantly, altered expression associated with the disease itself might modulate the transporter profile of target cells, thereby determining drug bioavailability and action. Drug transporter pharmacology has been periodically reviewed. Thus, with this contribution we aim at providing a state-of-the-art overview of the clinical evidence generated so far supporting the concept that these membrane proteins can indeed be biomarkers suitable for diagnosis and/or prognosis. Last but not least, some of these transporter proteins can also be envisaged as drug targets, as long as they can show “transceptor” functions, in some cases related to their role as modulators of extracellular adenosine levels, thereby providing a functional link between P1 receptors and transporters.

Synthesis of purine and 7-deazapurine nucleoside analogues of 6-N-(4-Nitrobenzyl)adenosine; inhibition of nucleoside transport and proliferation of cancer cells.

Human equilibrative nucleoside transporter 1 (hENT1) is a prototypical nucleoside transporter protein ubiquitously expressed on the cell surface of almost all human tissue. Given the role of hENT1 in the transport of nucleoside drugs, an important class of therapeutics in the treatment of various cancers and viral infections, efforts have been made to better understand the mechanisms by which hENT1 modulates nucleoside transport. To that end, we report here the design and synthesis of novel tool compounds for the further study of hENT1. The 7-deazapurine nucleoside antibiotic tubercidin was converted into its 4-N-benzyl and 4-N-(4-nitrobenzyl) derivatives by alkylation at N3 followed by a Dimroth rearrangement to the 4-N-isomer or by fluoro-diazotization followed by SN Ar displacement of the 4-fluoro group by a benzylamine. The 4-N-(4-nitrobenzyl) derivatives of sangivamycin and toyocamycin antibiotics were prepared by the alkylation approach. Cross-membrane transport of labeled uridine by hENT1 was inhibited to a weaker extent by the 4-nitrobenzylated tubercidin and sangivamycin analogues than was observed with 6-N-(4-nitrobenzyl)adenosine. Type-specific inhibition of cancer cell proliferation was observed at micromolar concentrations with the 4-N-(4-nitrobenzyl) derivatives of sangivamycin and toyocamycin, and also with 4-N-benzyltubercidin. Treatment of 2',3',5'-O-acetyladenosine with aryl isocyanates gave the 6-ureido derivatives but none of them exhibited inhibitory activity against cancer cell proliferation or hENT1.