Effects of gefitinib and vandetanib on human equilibrative nucleoside transporter 1 and on gemcitabine cytotoxicity.

2546 Background: Combination chemotherapy with tyrosine kinase inhibitors (TKIs) and gemcitabine has been attempted with little added benefit to patients. We hypothesized that TKIs that were designed to bind to ATP pockets of growth factor tyrosine kinases also bind to proteins that recognize nucleosides, thereby potentially interfering with gemcitabine pharmacology. Methods: Interaction of TKIs with human nucleoside transporters (NTs) was studied using recombinant NTs produced in yeast. Effects of TKIs on uridine transport, gemcitabine transport and accumulation, regulation of NT activity and cytotoxicity with and without gemcitabine were evaluated in human A549 lung cancer cells. Results: In yeast, vandetanib inhibited two equilibrative NTs (hENT1, hENT2) and three concentrative NTs (hCNT1, hCNT2, hCNT3) with the greatest inhibition seen with hENT1 whereas gefitinib strongly inhibited hENT1 and hCNT1 only. In A549 cells, which possess major hENT1 and minor hENT2 activities, [3H]uridine uptake was inhibited by vandetanib and gefitinib with IC50 values of 16 ± 4 and 5 ± 0.3µM, respectively. Both TKIs also inhibited [3H]gemcitabine transport and accumulation in A549 cells. hENT1 protein levels were decreased during exposures to vandetanib or gefitinib for 24 hours, and cytotoxicity was greatest when gemcitabine was given prior to vandetanib or gefitinib. Conclusions: Vandetanib and gefitinib inhibited human NTs, especially hENT1, resulting in reduced intracellular gemcitabine accumulation. Gefitinib or vandetanib levels achieved in plasma and tumor tissues are sufficient to inhibit hENT1 activity. Because TKIs can block uptake of nucleoside chemotherapy drugs in cultured cancer cells, attention must be paid to TKIs and nucleoside pharmacokinetic properties when scheduling TKIs and nucleoside chemotherapy.

Direct Sorting of the Yeast Uracil Permease to the Endosomal System Is Controlled by Uracil Binding and Rsp5p-dependent Ubiquitylation

The yeast uracil permease, Fur4p, is downregulated by uracil, which is toxic to cells with high permease activity. Uracil promotes cell surface Rsp5p-dependent ubiquitylation of the permease, signaling its endocytosis and further vacuolar degradation. We show here that uracil also triggers the direct routing of its cognate permease from the Golgi apparatus to the endosomal system for degradation, without passage via the plasma membrane. This early sorting was not observed for a variant permease with a much lower affinity for uracil, suggesting that uracil binding is the signal for the diverted pathway. The FUI1-encoded uridine permease is similarly sorted for early vacuolar degradation in cells exposed to a toxic level of uridine uptake. Membrane proteins destined for vacuolar degradation require sorting at the endosome level to the intraluminal vesicles of the multivesicular bodies. In cells with low levels of Rsp5p, Fur4p can be still diverted from the Golgi apparatus but does not reach the vacuolar lumen, being instead missorted to the vacuolar membrane. Correct luminal delivery is restored by the biosynthetic addition of a single ubiquitin, suggesting that the ubiquitylation of Fur4p serves as a specific signal for sorting to the luminal vesicles of the multivesicular bodies. A fused ubiquitin is also able to sort some Fur4p from the Golgi to the degradative pathway in the absence of added uracil but the low efficiency of this sorting indicates that ubiquitin does not itself act as a dominant signal for Golgi-to-endosome trafficking. Our results are consistent with a model in which the binding of intracellular uracil to the permease signals its sorting from the Golgi apparatus and subsequent ubiquitylation ensures its delivery to the vacuolar lumen.

Prolactin, Cortisol, and Insulin Regulation of Nucleoside Uptake Into Mouse Mammary Gland Explants

Nucleosides are essential components of milk that are used for the nourishment of newborns. Effects of the three primary lactogenic hormones, including prolactin (PRL), insulin (I), and cortisol (H), on nucleoside uptake and incorporation into cultured mammary tissues taken from 12- to 14-day pregnant mice were determined; most experiments focused on the regulation of uridine uptake. Insulin alone, as well as PRL in the presence of insulin and cortisol, was shown to stimulate uridine uptake and incorporation into RNA in mammary explants taken from 12- to 14-day pregnant mice. The PRL effects were expressed at concentrations of 25 ng/ml and above, which are physiological plasma concentrations. In the absence of sodium, uridine uptake and incorporation were diminished, suggesting the presence of a sodium-dependent uridine transporter. In kinetic studies the apparent Km for uridine uptake was calculated to be 312 μM, and the Vmax 2.90 μmol/hr/L cell water; PRL had no effect on the Km but increased the Vmax to 5.88 μmol/hr/L cell water. When assessing uridine uptake in the presence of the other nucleosides at 0.1 mM, only cytidine competed with uridine uptake. The fact that distribution ratios of greater than 15:1 were achieved with uridine indicates that uridine uptake may be via an active transporter. These studies show that PRL enhances uridine update in mammary tissues by stimulating the activity, and probably synthesis, of a sodium-dependent, active uridine and cytosine transporter.

Two Nucleoside Uptake Systems in Lactococcus lactis: Competition between Purine Nucleosides and Cytidine Allows for Modulation of Intracellular Nucleotide Pools

ABSTRACT A method for measuring internal nucleoside triphosphate pools of lactococci was optimized and validated. This method is based on extraction of 33P-labeled nucleotides with formic acid and evaluation by two-dimensional chromatography with a phosphate buffer system for the first dimension and with an H3BO3-LiOH buffer for separation in the second dimension. We report here the sizes of the ribo- and deoxyribonucleotide pools in laboratory strain MG1363 during growth in a defined medium. We found that purine- and pyrimidine-requiring strains may be used to establish physiological conditions in batch fermentations with altered nucleotide pools and growth rates by addition of nucleosides in different combinations. Addition of cytidine together with inosine to a purine-requiring strain leads to a reduction in the internal purine nucleotide pools and a decreased growth rate. This effect was not seen if cytidine was replaced by uridine. A similar effect was observed if cytidine and inosine were added to a pyrimidine-requiring strain; the UTP pool size was significantly decreased, and the growth rate was reduced. To explain the observed inhibition, the nucleoside transport systems in Lactococcus lactis were investigated by measuring the uptake of radioactively labeled nucleosides. The Km for for inosine, cytidine, and uridine was determined to be in the micromolar range. Furthermore, it was found that cytidine and inosine are competitive inhibitors of each other, whereas no competition was found between uridine and either cytidine or inosine. These findings suggest that there are two different high-affinity nucleoside transporters, one system responsible for uridine uptake and another system responsible for the uptake of all purine nucleosides and cytidine.

An ancient prevertebrate Na+-nucleoside cotransporter (hfCNT) from the Pacific hagfish (Eptatretus stouti)

The human concentrative (Na+-linked) plasma membrane transport proteins hCNT1, hCNT2, and hCNT3 are pyrimidine nucleoside-selective (system cit), purine nucleoside-selective (system cif), or broadly selective for both pyrimidine and purine nucleosides (system cib), respectively. All have orthologs in other mammalian species and belong to a gene family (CNT) that has members in insects, nematodes, pathogenic yeast, and bacteria. Here, we report the cDNA cloning and functional characterization of a CNT family member from an ancient marine prevertebrate, the Pacific hagfish ( Eptatretus stouti). This Na+-nucleoside symporter, designated hfCNT, is the first transport protein to be characterized in detail in hagfish and is a 683-amino acid residue protein with 13 predicted transmembrane helical segments (TMs). hfCNT was 52, 50, and 57% identical in sequence to hCNT1, hCNT2, and hCNT3, respectively. Similarity to hCNT3 was particularly marked in the TM 4–13 region. When produced in Xenopus oocytes, hfCNT exhibited the transport properties of system cib, with uridine, thymidine, and inosine apparent K m values of 10–45 μM. The antiviral nucleoside drugs 3′-azido-3′-deoxythymidine, 2′,3′-dideoxycytidine, and 2′,3′-dideoxyinosine were also transported. Simultaneous measurement of uridine-evoked currents and radiolabeled uridine uptake under voltage-clamp conditions gave a Na+-to-uridine coupling ratio of 2:1 (cf. 2:1 for hCNT3 and 1:1 for hCNT1/2). The apparent K 50 value for Na+ activation was >100 mM. A 50:50 chimera between hfCNT and hCNT1 (TMs 7–13 of hfCNT replaced by those of hCNT1) exhibited hCNT1-like cation interactions, establishing that the structural determinants of cation stoichiometry and binding affinity were located within the carboxy-terminal half of the protein. The high degree of sequence similarity between hfCNT and hCNT3 may indicate functional constraints on the primary structure of the transporter and suggests that cib-type CNTs fulfill important physiological functions.

Characterization of nucleoside transport systems in cultured rat epididymal epithelium

The nucleoside transport systems in cultured epididymal epithelium were characterized and found to be similar between the proximal (caput and corpus) and distal (cauda) regions of the epididymis. Functional studies revealed that 70% of the total nucleoside uptake was Na+ dependent, while 30% was Na+ independent. The Na+-independent nucleoside transport was mediated by both the equilibrative nitrobenzylthioinosine (NBMPR)-sensitive system (40%) and the NBMPR-insensitive system (60%), which was supported by a biphasic dose response to NBMPR inhibition. The Na+-dependent [3H]uridine uptake was selectively inhibited 80% by purine nucleosides, indicating that the purine nucleoside-selective N1 system is predominant. Since Na+-dependent [3H]guanosine uptake was inhibited by thymidine by 20% and Na+-dependent [3H]thymidine uptake was broadly inhibited by purine and pyrimidine nucleosides, this suggested the presence of the broadly selective N3 system accounting for 20% of Na+-dependent nucleoside uptake. Results of RT-PCR confirmed the presence of mRNA for equilibrative nucleoside transporter (ENT) 1, ENT2, and concentrative nucleoside transporter (CNT) 2 and the absence of CNT1. It is suggested that the nucleoside transporters in epididymis may be important for sperm maturation by regulating the extracellular concentration of adenosine in epididymal plasma.