OBJECTIVE

Gemcitabine is widely used to treat non-small cell lung cancer (NSCLC). The aim of this study was to assess the pharmacogenomic effects of the entire gemcitabine metabolic pathway, we genotyped single nucleotide polymorphisms (SNPs) within the 17 pathway genes using DNA samples from patients with NSCLC treated with gemcitabine to determine the effect of genetic variants within gemcitabine pathway genes on overall survival (OS) of patients with NSCLC after treatment of gemcitabine.

METHODS

Eight of the 17 pathway genes were resequenced with DNA samples from Coriell lymphoblastoid cell lines (LCLs) using Sanger sequencing for all exons, exon-intron junctions, and 5'-, 3'-UTRs. A total of 107 tagging SNPs were selected on the basis of the resequencing data for the eight genes and on HapMap data for the remaining nine genes, followed by successful genotyping of 394 NSCLC patient DNA samples. Association of SNPs/haplotypes with OS was performed using the Cox regression model, followed by functional studies performed with LCLs and NSCLC cell lines.

RESULTS

Five SNPs in four genes (CDA, NT5C2, RRM1, and SLC29A1) showed associations with OS of those patients with NSCLC, as well as nine haplotypes in four genes (RRM1, RRM2, SLC28A3, and SLC29A1) with a P value of less than 0.05. Genotype imputation using the LCLs was performed for a region of 200 kb surrounding those SNPs, followed by association studies with gemcitabine cytotoxicity. Functional studies demonstrated that downregulation of SLC29A1, NT5C2, and RRM1 in NSCLC cell lines altered cell susceptibility to gemcitabine.

CONCLUSIONS

These studies help in identifying biomarkers to predict gemcitabine response in NSCLC, a step toward the individualized chemotherapy of lung cancer.

OBJECTIVE

The purpose of this study was to identify the major ribavirin uptake transporter(s) in human hepatocytes and to determine if these previously unidentified transporters are involved in hepatic ribavirin uptake. Furthermore, we aimed to address what causes the difference in uptake levels among human hepatocytes.

METHODS

Profiles of ribavirin uptake and nucleoside transporter mRNA expression in Caucasian hepatocytes (HH268, HH283 and HH291) were characterized by transport assay and reverse transcription-polymerase chain reaction (RT-PCR). The 5'-side of the SLC29A1 gene structure was characterized by determination of transcription start sites and by RT-PCR.

RESULTS

Equilibrative nucleoside transporter 1 (ENT1)-mediated uptake was exclusively involved in ribavirin uptake in HH268 and HH283 and was responsible for the largest ribavirin uptake fraction in HH291. The level of ENT1-mediated uptake in HH291 was higher than that in HH268 and HH283. Characterization of the SLC29A1 gene structure revealed the existence of several ENT1 mRNA isoforms in the human liver, and the levels of four ENT1 mRNA isoforms in HH291 were higher than those in HH268 or HH283. No ENT2-mediated uptake was observed in any hepatocyte lines. Na(+)-dependent uptake was detected only in HH291; however, mRNA levels of concentrative nucleoside transporters (CNTs) were at trace levels in all hepatocyte lines.

CONCLUSIONS

ENT1, but not ENT2 or CNTs, is a major ribavirin uptake transporter in human hepatocytes. The different ENT1-mediated ribavirin uptake levels in different hepatocyte lines are associated with different expression levels of specific isoforms of ENT1 mRNAs. Furthermore, an unidentified Na(+)-dependent ribavirin transport system might exist in human hepatocytes.

BACKGROUND

The comparison of organ transcriptomes is an important strategy for understanding gene functions. In the present study, we attempted to identify lung-prominent genes by comparing the normal transcriptomes of rat lung, heart, kidney, liver, spleen, and brain. To increase the efficiency and reproducibility, we first developed a novel parallel hybridization system, in which 6 samples could be hybridized onto a single slide at the same time.

RESULTS

We identified the genes prominently expressed in the lung (147) or co-expressed in lung-heart (23), lung-liver (37), lung-spleen (203), and lung-kidney (98). The known functions of the lung-prominent genes mainly fell into 5 categories: ligand binding, signal transducer, cell communication, development, and metabolism. Real-time PCR confirmed 13 lung-prominent genes, including 5 genes that have not been investigated in the lung, vitamin D-dependent calcium binding protein (Calb3), mitogen activated protein kinase 13 (Mapk13), solute carrier family 29 transporters, member 1 (Slc29a1), corticotropin releasing hormone receptor (Crhr1), and lipocalin 2 (Lcn2).

CONCLUSIONS

The lung-prominent genes identified in this study may provide an important clue for further investigation of pulmonary functions.

BACKGROUND

Nucleoside transporter proteins (NTs) encoded by members of the SLC28 and SLC29 gene families contribute to nucleoside and nucleobase recycling but also modulate extracellular adenosine levels and thus adenosine-regulated metabolic targets.

METHODS

We have examined the expression pattern of NT-encoding genes in human adipose tissue and we have further analysed whether the mRNA related to these genes show changes in their amounts associated with either HIV-1 infection, highly active antiretroviral therapy (HAART) or development of HIV-1-associated lipodystrophy syndrome (HALS).

RESULTS

Human adipocytes express SLC28A1, SLC28A2 and SLC28A3 (encoding hCNT1, hCNT2 and hCNT3, respectively) and SLC29A1 and SLC29A2 (encoding hENT1 and hENT2, respectively). HIV-1 infection, prior to HAART and HALS development, is associated with the upregulation of the mRNA levels of the genes encoding hCNT1, hCNT3 and hENT2. The increase in the mRNA amounts for the former two genes may be due to the action of tumour necrosis factor-alpha (TNF-alpha), a cytokine with enhanced expression in adipose tissue following HIV-1 infection, as the effect is also observed in human adipocytes in culture after treatment with TNF-alpha. HAART and HALS development are associated with the upregulation of the mRNA levels encoding hCNT2 and hENT1, and further enhancement of hCNT1, hCNT3 and hENT2 gene expression.

CONCLUSIONS

These data suggest that selected genes of the SLC28 and SLC29 families are not only targets of HIV-1 infection, but might also contribute to the development of adipose tissue alterations leading to lipodystrophy.

Two subtypes of equilibrative transporters, es (equilibrative inhibitor-sensitive) and ei (equilibrative inhibitor-insensitive), are responsible for the majority of nucleoside flux across mammalian cell membranes. Sequence analyses of the representative genes, ENT1 {equilibrative nucleoside transporter 1; also known as SLC29A1 [solute carrier family 29 (nucleoside transporters), member 1]} and ENT2 (SLC29A2), suggest that protein kinase CK2-mediated phosphorylation may be involved in the regulation of es- and ei-mediated nucleoside transport. We used human osteosarcoma cells transfected with catalytically active or inactive alpha' and alpha subunits of CK2 to assess the effects of CK2 manipulation on nucleoside transport activity. Expression of inactive CK2alpha' (decreased CK2alpha' activity) increased the number of binding sites (approximately 1.5-fold) for the es-specific probe [3H]NBMPR ([3H]nitrobenzylthioinosine), and increased (approximately 1.8-fold) the V(max) for 2-chloro[3H]adenosine of the NBMPR-sensitive (es) nucleoside transporter. There was a concomitant decrease in the V(max) of the NBMPR-resistant (ei-mediated) uptake of 2-chloro[3H]adenosine. This inhibition of CK2alpha' activity had no effect, however, on either the K(D) of [3H]NBMPR binding or the K(m) of 2-chloro[3H]adenosine uptake. Quantitative PCR showed a transient decrease in the expression of both hENT1 (human ENT1) and hENT2 mRNAs within 4-12 h of induction of the inactive CK2alpha' subunit, but both transcripts had returned to control levels by 24 h. These data suggest that inhibition of CK2alpha' reduced ei activity by attenuation of hENT2 transcription, while the increase in es/hENT1 activity was mediated by post-translational action of CK2. The observed modification in es activity was probably due to a CK2alpha'-mediated change in the phosphorylation state of the ENT1 protein, or an interacting protein, effecting an increase in the plasma membrane lifetime of the transport proteins.

Alternative splicing is a tightly regulated process whereby non-coding sequences of pre-mRNA are removed and protein-coding segments are assembled in diverse combinations, ultimately giving rise to proteins with distinct or even opposing functions. In the past decade, whole genome/transcriptome sequencing studies revealed the high complexity of splicing regulation, which occurs co-transcriptionally and is influenced by chromatin status and mRNA modifications. Consequently, splicing profiles of both healthy and malignant cells display high diversity and alternative splicing was shown to be widely deregulated in multiple cancer types. In particular, mutations in pre-mRNA regulatory sequences, splicing regulators and chromatin modifiers, as well as differential expression of splicing factors are important contributors to cancer pathogenesis. It has become clear that these aberrations contribute to many facets of cancer, including oncogenic transformation, cancer progression, response to anticancer drug treatment as well as resistance to therapy. In this respect, alternative splicing was shown to perturb the expression a broad spectrum of relevant genes involved in drug uptake/metabolism (i.e. SLC29A1, dCK, FPGS, and TP), activation of nuclear receptor pathways (i.e. GR, AR), regulation of apoptosis (i.e. MCL1, BCL-X, and FAS) and modulation of response to immunotherapy (CD19). Furthermore, aberrant splicing constitutes an important source of novel cancer biomarkers and the spliceosome machinery represents an attractive target for a novel and rapidly expanding class of therapeutic agents. Small molecule inhibitors targeting SF3B1 or splice factor kinases were highly cytotoxic against a wide range of cancer models, including drug-resistant cells. Importantly, these effects are enhanced in specific cancer subsets, such as splicing factor-mutated and c-MYC-driven tumors. Furthermore, pre-clinical studies report synergistic effects of spliceosome modulators in combination with conventional antitumor agents. These strategies based on the use of low dose splicing modulators could shift the therapeutic window towards decreased toxicity in healthy tissues. Here we provide an extensive overview of the latest findings in the field of regulation of splicing in cancer, including molecular mechanisms by which cancer cells harness alternative splicing to drive oncogenesis and evade anticancer drug treatment as well as splicing-based vulnerabilities that can provide novel treatment opportunities. Furthermore, we discuss current challenges arising from genome-wide detection and prediction methods of aberrant splicing, as well as unravelling functional relevance of the plethora of cancer-related splicing alterations.

Keywords: Alternative splicing; Cancer; Drug resistance; Oncogenesis; SF3B1 inhibition; Splicing factor mutation; Splicing modulation.

Multistep tumorigenesis is a form of microevolution consisting of mutation and selection. To clarify the role of selection modalities in tumor development, we examined two alternative evolutionary conditions, r-selection in sparse culture, which allows cells to proliferate rapidly, and K-selection in confluent culture, in which overcrowding constrains cell proliferation. Using MYC- and EJ-RAS-transformed rat embryo fibroblasts, we found that K-selected cells acquired and stably maintained multidrug resistance (MDR) to DOX, VCR, MTX and Ara-C. Then, we examined the involvement of a number of factors potentially causal of the development of MDR, that is, ploidy, Tp53 mutation, doubling time and the expression levels of genes related to drug resistance. Although ploidy status and Tp53 mutations did not correlate with MDR, we found that Abcb1/Mdr1, encoding P-glycoprotein (Pgp), was significantly upregulated after K-selection. Cyclosporin A, a competitive inhibitor of Pgp, increased the intracellular accumulation of DOX and reduced the resistance to it. Indeed, the population of Pgp-transfected cells significantly expanded under K-, but not under r-selection. In addition to Pgp upregulation, altered expression of other genes such as Cda/cytidine deaminase and Slc29a1/equilibrative nucleoside transporter 1 and prolonged doubling times were associated with MDR. This system reproduces events associated with MDR in vivo and would be useful for analysis of MDR development.

Human umbilical vein endothelial cells (HUVEC) from gestational diabetes exhibit reduced adenosine uptake and increased nitric oxide (NO) synthesis. Adenosine transport via human equilibrative nucleoside transporters 1 (hENT1) is reduced by NO by unknown mechanisms in HUVEC. We examined whether gestational diabetes-reduced adenosine transport results from lower hENT1 gene (SLC29A1) expression. HUVEC from gestational diabetes exhibit reduced SLC29A1 promoter activity when transfected with pGL3-hENT1(-2154) compared with pGL3-hENT1(-1114) constructs, an effect blocked by N(G)-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor), but unaltered by S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor). In cells from gestational diabetes transfected with pGL3-hENT1(-2154), L-NAME increased, but SNAP did not alter promoter activity and hENT1 expression. However, in cells from normal pregnancies L-NAME increased, but SNAP reduced promoter activity and hENT1 expression. Adenovirus-silenced eNOS expression increased hENT1 expression and activity in cells from normal or gestational diabetic pregnancies. Thus, reduced adenosine transport may result from downregulation of SLC29A1 expression by NO in HUVEC from gestational diabetes. These findings explain the accumulation of extracellular adenosine detected in cultures of HUVEC from gestational diabetes. In addition, fetal endothelial dysfunction could be involved in the abnormal fetal development and growth seen in gestational diabetes.

High D-glucose reduces human equilibrative nucleoside transporter 1 (hENT1)-mediated adenosine uptake involving endothelial nitric oxide synthase (eNOS), mitogen-activated protein (MAP) kinase kinases 1 and 2/MAP kinases p42/44 (MEK/ERKs), and protein kinase C (PKC) activation in human umbilical vein endothelium (HUVEC). Since NO represses SLC29A1 gene (hENT1) promoter activity we studied whether D-glucose-reduced hENT1-adenosine transport results from lower SLC29A1 expression in HUVEC primary cultures. HUVEC incubation (24 h) with high D-glucose (25 mM) reduced hENT1-adenosine transport and pGL3-hENT1(-1114) construct SLC29A1 reporter activity compared with normal D-glucose (5 mM). High D-glucose also reduced pGL3-hENT1(-1114) reporter activity compared with cells transfected with pGL3-hENT1(-795) construct. N(G)-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor), PD-98059 (MEK1/2 inhibitor), and/or calphostin C (PKC inhibitor) blocked D-glucose effects. Insulin (1 nM) and phorbol 12-myristate 13-acetate (PMA, 100 nM, PKC activator), but not 4alpha-phorbol 12,13-didecanoate (4alphaPDD, 100 nM, PMA less active analogue) reduced hENT1-adenosine transport. L-NAME and PD-98059 blocked insulin effects. L-NAME, PD-98059, and calphostin C increased hENT1 expression without altering protein or mRNA stability. High D-glucose increased Sp1 transcription factor protein abundance and binding to SLC29A1 promoter, phenomena blocked by L-NAME, PD-98059, and calphostin C. Sp1 overexpression reduced SLC29A1 promoter activity in normal D-glucose, an effect reversed by L-NAME and further reduced by S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor) in high D-glucose. Thus, reduced hENT1-mediated adenosine transport in high D-glucose may result from increased Sp1 binding to SLC29A1 promoter down-regulating hENT1 expression. This phenomenon depends on eNOS, MEK/ERKs, and PKC activity, suggesting potential roles for these molecules in hyperglycemia-associated endothelial dysfunction.

OBJECTIVE

The standard of hepatitis C antiviral therapy combines pegylated interferon-α with ribavirin. This polar guanosine analog improves the sustained virological response (SVR) rates, but may induce hemolytic anemia. As its pharmacokinetics depend on facilitated transmembrane transport, we assessed whether variants in genes that code for concentrative (concentrative nucleoside transporters 2 and 3 coded by SLC28A2 and SLC28A3, respectively) and equilibrative nucleoside transporters (equilibrative nucleoside transporters 1 and 2 coded by SLC29A1 and SLC29A2, respectively) are associated with the therapy response and side effects.

METHODS

Patients (n=169) chronically infected with the hepatitis C virus genotype 1, treated with standard doses of pegylated interferon-α and weight-based doses of ribavirin for up to 48 weeks, were genotyped for 21 variants in nucleoside transporter genes SLC28A2, SLC28A3, SLC29A1, and SLC29A2, selected to include reported functional variants and to span the complete gene loci. The presence or absence of a SVR (n=169) and a relevant decrease (>3 g/dl, n=115) in blood hemoglobin were associated with the genotypes.

RESULTS

The variant SLC28A3 haplotype rs10868138G/rs56350726T (allelic frequency 0.074) was associated with a lower incidence (35.5%) of relevant decreases (>3 g/dl) in blood hemoglobin than in noncarriers (64.3%; P=0.024, n=115). This protection against hemolytic anemia was not associated with decreased SVR rates (n=169).

CONCLUSIONS

A genetic variant in SCL28A3 coding for the concentrative nucleoside transporter 3 protects patients with chronic hepatitis C against hemolytic anemia without affecting SVR in hepatitis C virus genotype 1.

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm resulting from the malignant transformation of myeloid progenitors. Despite intensive chemotherapy leading to initial treatment responses, relapse caused by intrinsic or acquired drug resistance represents a major challenge. Here, we report that histone 3 lysine 27 demethylase KDM6A (UTX) is targeted by inactivating mutations and mutation-independent regulation in relapsed AML. Analyses of matched diagnosis and relapse specimens from individuals with KDM6A mutations showed an outgrowth of the KDM6A mutated tumor population at relapse. KDM6A expression is heterogeneously regulated and relapse-specific loss of KDM6A was observed in 45.7% of CN-AML patients. KDM6A-null myeloid leukemia cells were more resistant to treatment with the chemotherapeutic agents cytarabine (AraC) and daunorubicin. Inducible re-expression of KDM6A in KDM6A-null cell lines suppressed proliferation and sensitized cells again to AraC treatment. RNA expression analysis and functional studies revealed that resistance to AraC was conferred by downregulation of the nucleoside membrane transporter ENT1 (SLC29A1) by reduced H3K27 acetylation at the ENT1 locus. Our results show that loss of KDM6A provides cells with a selective advantage during chemotherapy, which ultimately leads to the observed outgrowth of clones with KDM6A mutations or reduced KDM6A expression at relapse.

Inhibitor and substrate interactions with equilibrative nucleoside transporter 1 (ENT1; SLC29A1) are known to be affected by cysteine-modifying reagents. Given that selective ENT1 inhibitors, such as nitrobenzylmercaptopurine riboside (NBMPR), bind to the N-terminal half of the ENT1 protein, we hypothesized that one or more of the four cysteine residues in this region were contributing to the effects of the sulfhydryl modifiers. Recombinant human ENT1 (hENT1), and the four cysteine-serine ENT1 mutants, were expressed in nucleoside transport-deficient PK15 cells and probed with a series of methanethiosulfonate (MTS) sulfhydryl-modifying reagents. Transporter function was assessed by the binding of [(3)H]NBMPR and the cellular uptake of [(3)H]2-chloroadenosine. The membrane-permeable reagent methyl methanethiosulfonate (MMTS) enhanced [(3)H]NBMPR binding in a pH-dependent manner, but decreased [(3)H]2-chloroadenosine uptake. [2-(Trimethylammonium)ethyl] methane-thiosulfonate (MTSET) (positively charged, membrane-impermeable), but not sodium (2-sulfonatoethyl)-methanethiosulfonate (MTSES) (negatively charged), inhibited [(3)H]NBMPR binding and enhanced [(3)H]2-chloroadenosine uptake. Mutation of Cys222 in transmembrane (TM) 6 eliminated the effect of MMTS on NBMPR binding. Mutation of Cys193 in TM5 enhanced the ability of MMTS to increase [(3)H]NBMPR binding and attenuated the effects of MMTS and MTSET on [(3)H]2-chloroadenosine uptake. Taken together, these data suggest that Cys222 contributes to the effects of MTS reagents on [(3)H]NBMPR binding, and Cys193 is involved in the effects of these reagents on [(3)H]2-chloroadenosine transport. The results of this study also indicate that the hENT1-C193S mutant may be useful as a MTSET/MTSES-insensitive transporter for future cysteine substitution studies to define the extracellular domains contributing to the binding of substrates and inhibitors to this critical membrane transporter.

Membrane drug transporters contribute to the disposition of many drugs. In human liver, drug transport is controlled by two main superfamilies of transporters, the solute carrier transporters (SLC) and the ATP Binding Cassette transporters (ABC). Altered expression of these transporters due to drug-drug interactions can contribute to differences in drug exposure and possibly effect. In this study, we determined the effect of rifampin on gene expression of hundreds of membrane transporters along with all clinically relevant drug transporters.

METHODS

In this study, primary human hepatocytes (n = 7 donors) were cultured and treated for 24 h with rifampin and vehicle control. RNA was isolated from the hepatocytes, mRNA expression was measured by RNA-seq, and miRNA expression was analyzed by Taqman OpenArray. The effect of rifampin on the expression of selected transporters was also tested in kidney cell lines. The impact of rifampin on the expression of 410 transporter genes from 19 different transporter gene families was compared with vehicle control.

RESULTS

Expression patterns of 12 clinically relevant drug transporter genes were changed by rifampin (FDR < 0.05). For example, the expressions of ABCC2, ABCB1, and ABCC3 were increased 1.9-, 1.7-, and 1.2-fold, respectively. The effects of rifampin on four uptake drug transporters (SLCO1B3, SLC47A1, SLC29A1, SLC22A9) were negatively correlated with the rifampin effects on specific microRNA expression (SLCO1B3/miR-92a, SLC47A1/miR-95, SLC29A1/miR-30d#, and SLC22A9/miR-20; r < -0.79; p < 0.05). Seven hepatic drug transporter genes (SLC22A1, SLC22A5, SLC15A1, SLC29A1, SLCO4C1, ABCC2, and ABCC4), whose expression was altered by rifampin in hepatocytes, were also present in a renal proximal tubular cell line, but in renal cells rifampin did not alter their gene expression. PXR expression was very low in the kidney cells; this may explain why rifampin induces gene expression in a tissue-specific manner.

CONCLUSIONS

Rifampin alters the expression of many of the clinically relevant hepatic drug transporters, which may provide a rational basis for understanding rifampin-induced drug-drug interactions reported in vivo. The relevance of its effect on many other transporters remains to be studied.

The liver plays a central role in allowing dairy cattle to make a successful transition into lactation. In liver, as in other tissues, extracellular nucleotides and nucleosides trigger cellular responses through adenosine and ATP receptors. Adenosine triphosphate and certain nucleotides serve as signals that can heighten purinergic receptor activation in several pathologic processes. We evaluated the mRNA expression of genes associated with the purinergic signaling network in liver tissue during the peripartal period. Seven multiparous Holstein cows were dried off at d -50 relative to expected parturition and fed a controlled-energy diet (net energy for lactation=1.24 Mcal/kg of DM) for ad libitum intake during the entire dry period. After calving, all cows were fed a common lactation diet (net energy for lactation=1.65 Mcal/kg of DM) until 30 DIM. Biopsies of liver were harvested at d -10, 7, and 21 for mRNA expression of 9 purinergic receptors, 7 ATP and adenosine transport channels, and 10 enzymes associated with ATP hydrolysis. Blood collected at d -21, -10, 7, 14, and 21 was used to measure concentrations of inflammation and oxidative stress biomarkers. The expression of type 1 purinergic receptors (ADORA2A and ADORA3), several nucleoside hydrolases [ectonucleoside triphosphate diphosphohydrolase 7 (ENTPD7), ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), ENPP3, and adenosine deaminase (ADA)], and a type 2 purinergic receptor (P2RX7) was downregulated after calving. In contrast, the expression of type 2 purinergic receptors (P2RX4 and PR2Y11), an ATP release channel (gap junction hemichannel GJB1), and an adenosine uptake protein (SLC29A1) followed the opposite response, increasing after calving and remaining elevated through 21 d. Haptoglobin, ceruloplasmin, and reactive oxygen metabolite concentrations increased gradually from d -21 d through at least d 7. The opposite response was observed for albumin, paraoxonase, α-tocopherol, and nitric oxide, which decreased gradually to a nadir at 7 and 14 d. Our results suggest that alterations after calving of the expression of hepatic purinergic signaling genes could be functionally important because in nonruminants, they play roles in bile formation, glucose metabolism, cholesterol uptake, inflammation, and steatosis. The correlation analysis provided evidence of a link between purinergic signaling genes and biomarkers of inflammation and oxidative stress.

The weak base antipsychotic clozapine is the most effective medication for treating refractory schizophrenia. The brain-to-plasma concentration of unbound clozapine is greater than unity, indicating transporter-mediated uptake, which has been insufficiently studied. This is important, because it could have a significant impact on clozapine's efficacy, drug-drug interaction, and safety profile. A major limitation of clozapine's use is the risk of clozapine-induced agranulocytosis/granulocytopenia (CIAG), which is a rare but severe hematological adverse drug reaction. We first studied the uptake of clozapine into human brain endothelial cells (hCMEC/D3). Clozapine uptake into cells was consistent with a carrier-mediated process, which was time-dependent and saturable ( Vmax = 3299 pmol/million cells/min, Km = 35.9 μM). The chemical inhibitors lamotrigine, quetiapine, olanzapine, prazosin, verapamil, indatraline, and chlorpromazine reduced the uptake of clozapine by up to 95%. This could in part explain the in vivo interactions observed in rodents or humans for these compounds. An extensive set of studies utilizing transporter-overexpressing cell lines and siRNA-mediated transporter knockdown in hCMEC/D3 cells showed that clozapine was not a substrate of OCT1 (SLC22A1), OCT3 (SLC22A3), OCTN1 (SLC22A4), OCTN2 (SLC22A5), ENT1 (SLC29A1), ENT2 (SLC29A2), and ENT4/PMAT (SLC29A4). In a recent genome-wide analysis, the hepatic uptake transporters SLCO1B1 (OATP1B1) and SLCO1B3 (OATP1B3) were identified as additional candidate transporters. We therefore also investigated clozapine transport into OATP1B-transfected cells and found that clozapine was neither a substrate nor an inhibitor of OATP1B1 and OATP1B3. In summary, we have identified a carrier-mediated process for clozapine uptake into brain, which may be partly responsible for clozapine's high unbound accumulation in the brain and its drug-drug interaction profile. Cellular clozapine uptake is independent from currently known drug transporters, and thus, molecular identification of the clozapine transporter will help to understand clozapine's efficacy and safety profile.

The initial goal of this study was to investigate alterations in adenosine A2A receptor (A2AR) density or function in a rat model of Huntington disease (HD) with reported insensitivity to an A2AR antagonist. Unsuspected negative results led to the hypothesis of a low striatal adenosine tone and to the search for the mechanisms involved. Extracellular striatal concentrations of adenosine were measured with in vivo microdialysis in two rodent models of early neuropathological stages of HD disease, the Tg51 rat and the zQ175 knock-in mouse. In view of the crucial role of the equilibrative nucleoside transporter (ENT1) in determining extracellular content of adenosine, the binding properties of the ENT1 inhibitor [3H]-S-(4-Nitrobenzyl)-6-thioinosine were evaluated in zQ175 mice and the differential expression and differential coexpression patterns of the ENT1 gene (SLC29A1) were analyzed in a large human cohort of HD disease and controls. Extracellular striatal levels of adenosine were significantly lower in both animal models as compared with control littermates and striatal ENT1 binding sites were significantly upregulated in zQ175 mice. ENT1 transcript was significantly upregulated in HD disease patients at an early neuropathological severity stage, but not those with a higher severity stage, relative to non-demented controls. ENT1 transcript was differentially coexpressed (gained correlations) with several other genes in HD disease subjects compared to the control group. The present study demonstrates that ENT1 and adenosine constitute biomarkers of the initial stages of neurodegeneration in HD disease and also predicts that ENT1 could constitute a new therapeutic target to delay the progression of the disease.

OBJECTIVE

We investigated the associations between variants in genes coding for enzymes and transporters related to the 6-mercaptopurine pathway and clinical outcomes in pediatric patients with acute lymphoblastic leukemia.

METHODS

Statistical association between gender, age and genotypes of selected SNPs, and the risks of hematological toxicity and relapse were investigated using a Cox proportional hazard model in 70 acute lymphoblastic leukemia patients from upper Egypt.

RESULTS

We found significant associations between ITPA, IMPDH1, SLC29A1, SLC28A2, SLC28A3 and ABCC4 SNPs and one or more of the hematological toxicity manifestations (neutropenia, agranulocytosis and leukopenia); age was significantly related to relapse.

CONCLUSIONS

Genetic polymorphisms in enzymes and transporters involved in the 6-mercaptopurine pathway should be considered during its use to avoid hematological toxicity.

OBJECTIVE

The aim of this study was to evaluate the association of gemcitabine pathway SNPs with detailed pharmacokinetic measures obtained from solid tumor patients receiving gemcitabine-based therapy.

METHODS

SNPs within nine gemcitabine pathway genes, namely CDA, CMPK, DCK, DCTD, NT5C2, NT5C3, SLC28A1, SLC28A3 and SLC29A1 were analyzed for association with gemcitabine pharmacokinetics.

RESULTS

Significant association of gemcitabine clearance with SNPs in NT5C2 was identified. Clearance of 2´,2´-difluorodeoxyuridine, a gemcitabine metabolite was significantly predicted by CDA, SLC29A1 and NT5C2 SNPs. This study reports an association of formation clearance of 2´,2´-difluoro-2´-deoxycytidine triphosphate, an active form of gemcitabine with SNPs within uptake transporters SLC28A1, SLC28A3 and SLC29A1.

CONCLUSIONS

Genetic variation in gemcitabine pathway genes is associated with its pharmacokinetics and hence could influence gemcitabine response. Our study identified pharmacogenetic markers that could be further tested in larger patient cohorts and could open up opportunities to individualize therapy in solid tumor patients.

Cytarabine arabinoside (ara-C) is the key agent for treating acute myeloid leukaemia (AML). Here, we genotyped 139 single nucleotide polymorphisms (SNPs) within the ara-C transport and metabolic pathway using the Illumina Golden Gate Assay in 97 patients with previously non-treated de novo AML other than M3. DCK rs4694362 (CC genotype) was a significant poor prognostic factor for overall survival (OS) (hazard ratio [HR], 33.202 [95% confidence interval (CI), 4.937-223.273], P<0.0001, P(Bonferroni)=0.017). SLC29A1 rs3734703 (AA or AC genotype) in combination with TYMS rs2612100 (AA genotype) was significantly associated with shorter relapse free survival (RFS) (HR, 17.630 [95% CI, 4.829-64.369], P<0.0001, P(Bonferroni)=0.021). These SNPs showed moderate or large inter ethnic divergence in allele frequencies from African or Caucasian populations. The results of our study suggest that a single SNP and SNP-SNP interactions may help to predict the drug response and provide a guide in developing individualised chemotherapy for AML patients receiving ara-C based chemotherapy.

Trifluridine (FTD) is a key component of the novel oral antitumor drug TAS-102 (also named TFTD), which consists of FTD and a thymidine phosphorylase inhibitor. FTD is supposed to exert its cytotoxicity via massive misincorporation into DNA, but the underlying mechanism of FTD incorporation into DNA and its correlation with cytotoxicity are not fully understood. The present study shows that several antibodies against 5-bromo-2'-deoxyuridine (BrdU) specifically cross-react with FTD, either anchored to bovine serum albumin or incorporated into DNA. These antibodies are useful for several biological applications, such as fluorescence-activated cell sorting, fluorescent immunostaining and immunogold detection for electron microscopy. These techniques confirmed that FTD is mainly incorporated in the nucleus during S phase in a concentration-dependent manner. In addition, FTD was also detected by immunohistochemical staining in paraffin-embedded HCT-116 xenograft tumors after intraperitoneal administration of FTD. Intriguingly, FTD was hardly detected in surrounding matrices, which consisted of fibroblasts with marginal expression of the nucleoside transporter genes SLC29A1 and SLC29A2. Thus, applications using anti-BrdU antibodies will provide powerful tools to unveil the underlying mechanism of FTD action and to predict or evaluate the efficacy and adverse effects of TAS-102 clinically.

Although systemic chemotherapy significantly improves the overall survival of pancreatic cancer patients, the prognosis remains extremely poor. The development of a drug resistance, either de novo or induced resistance, significantly limits the effectiveness of chemotherapy. SLC29A1 gene encodes human equilibrative nucleoside transporter 1 (hENT1) protein that is mediating the transport of nucleotides, both purines and pyrimidines, into the tumor cells. The aim of this mini-review is to summarize the current information concerning the prognostic and predictive role of SLC29A1 transporter (hENT1) expression in pancreatic cancer. Increased expression of SLC29A1 in vitro has been described as a potential critical factor determining the sensitivity of pancreatic cancer cells to gemcitabine and 5-fluorouracil, the principal cytotoxic agents used in the treatment of pancreatic cancer. The reports on the relationship between SLC29A1 expression and prognosis of patients with pancreatic cancer are currently rather conflicting. However, majority of studies on patients with resected pancreatic cancer have suggested that high SLC29A1expression may be predictive of improved survival in patients treated with gemcitabine. SLC29A1 has not been shown to represent a predictive biomarker for patients treated by 5-fluorouracil. In conclusion, potential prognostic and predictive role of SLC29A1 has been demonstrated for selected subset of patients.

In the last decade it has been recapitulated that receptor-ligand binding kinetics is a relevant additional parameter in drug discovery to improve in vivo drug efficacy and safety. The equilibrative nucleoside transporter-1 (ENT1, SLC29A1) is an important drug target, as transporter inhibition is a potential treatment of ischemic heart disease, stroke, and cancer. Currently, two non-selective ENT1 inhibitors (dilazep and dipyridamole) are on the market as vasodilators. However, their binding kinetics are unknown; moreover, novel, more effective and selective inhibitors are still needed. Hence, this study focused on the incorporation of binding kinetics for finding new and improved ENT1 inhibitors. We developed a radioligand competition association assay to determine the binding kinetics of ENT1 inhibitors with four chemical scaffolds (including dilazep and dipyridamole). The kinetic parameters were compared to the affinities obtained from a radioligand displacement assay. Three of the scaffolds presented high affinities with relatively fast dissociation kinetics, yielding short to moderate residence times (RTs) at the protein (1-44 min). While compounds from the fourth scaffold, i.e. draflazine analogues, also had high affinity, they displayed significantly longer RTs, with one analogue (4) having a RT of over 10 h. Finally, a label-free assay was used to evaluate the impact of divergent ENT1 inhibitor binding kinetics in a functional assay. It was shown that the potency of compound 4 increased with longer incubation times, which was not observed for draflazine, supporting the importance of long RT for increased target-occupancy and effect. In conclusion, our research shows that high affinity ENT1 inhibitors show a large variation in residence times at this transport protein. As a consequence, incorporation of binding kinetic parameters adds to the design criteria and may thus result in a different lead compound selection. Taken together, this kinetic approach could inspire future drug discovery in the field of ENT1 and membrane transport proteins in general.

Transporters are important therapeutic but yet understudied targets due to lack of available assays. Here we describe a novel label-free, whole-cell method for the functional assessment of Solute Carrier (SLC) inhibitors. As many SLC substrates are also ligands for G protein-coupled receptors (GPCRs), transporter inhibition may affect GPCR signalling due to a change in extracellular concentration of the substrate/ligand, which can be monitored by an impedance-based label-free assay. For this study, a prototypical SLC/GPCR pair was selected, i.e. the equilibrative nucleoside transporter-1 (SLC29A1/ENT1) and an adenosine receptor (AR), for which adenosine is the substrate/ligand. ENT1 inhibition with three reference compounds was monitored sensitively via AR activation on human osteosarcoma cells. Firstly, the inhibitor addition resulted in an increased apparent potency of adenosine. Secondly, all inhibitors concentration-dependently increased the extracellular adenosine concentration, resulting in an indirect quantitative assessment of their potencies. Additionally, AR activation was abolished by AR antagonists, confirming that the monitored impedance was AR-mediated. In summary, we developed a novel assay as an in vitro model system that reliably assessed the potency of SLC29A1 inhibitors via AR signalling. As such, the method may be applied broadly as it has the potential to study a multitude of SLCs via concomitant GPCR signalling.