Expression of high levels of nitrobenzylthioinosine-sensitive nucleoside transport in cultured human choriocarcinoma (BeWo) cells

Boumah, Christine E.; Hogue, Douglas L.; Cass, Carol E.

doi:10.1042/bj2880987

Cited by 44 publications

(51 citation statements)

References 37 publications

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“…2B, Table II) exhibited K d and B max values of 0.9 Ϯ 0.2 nM and 75.0 Ϯ 5 pmol/mg of protein, respectively (mean Ϯ S.E., n ϭ 4). The K d value was essentially the same as that determined previously (0.6 nM) for the high affinity binding sites in intact BeWo cells (19), and there was no indication of a second set of lower affinity binding sites. There was a nearly 4-fold increase in specific binding activity relative to that of the crude membrane preparations ( Fig.…”

Section: Resultssupporting

confidence: 69%

“…BeWo Cells Express hENT1 and hENT2 mRNA-BeWo cells have previously been shown (19) to exhibit es and ei functional transport activity. Since hENT1 and hENT2 were initially identified by molecular cloning of a placental cDNA library (10,16) and the BeWo cell line is of placental origin (18), BeWo RNA was examined for evidence of expression of hENT1 and hENT2 mRNA.…”

Section: Resultsmentioning

confidence: 99%

“…BeWo membrane fractions collected at different sucrose concentrations were first assayed for NBMPR binding activity at a single concentration (5 nM) under conditions previously shown in studies with intact cells (19) to be sufficient to label over 40% of the high affinity sites. Crude membranes (the "postnuclear" supernatant) prepared from ϳ1 ϫ 10 9 cells bound 6.5 pmol of NBMPR/mg of protein when incubated with 5 nM NBMPR for 45 min at room temperature.…”

Section: Resultsmentioning

confidence: 99%

“…Previous studies with proliferating BeWo cells (19) (20,21), cultured leukemia (CEM) cells (22), and Ehrlich ascites tumor cells (23). The existence of two classes of binding sites, together with the lack of proportionality between NBMPR-binding sites and maximal transport activities of BeWo and HeLa cells, led to the suggestion (19) that the second class of NBMPR-binding sites in BeWo cells may be es transporters associated with intracellular membranes.…”

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confidence: 99%

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Demonstration of Equilibrative Nucleoside Transporters (hENT1 and hENT2) in Nuclear Envelopes of Cultured Human Choriocarcinoma (BeWo) Cells by Functional Reconstitution in Proteoliposomes

Mani¹,

Hammond²,

Marjan³

et al. 1998

Journal of Biological Chemistry

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Multiple nucleoside transport (NT)1 -mediated processes exist in mammalian cells and are divided into two groups, depending on whether they are equilibrative or concentrative in nature (for recent reviews, see Refs. 1-3). The equilibrative transporters accept both purine and pyrimidine nucleosides as permeants and are found in most, possibly all, cell types, whereas the concentrative transporters have relatively narrow selectivities for nucleoside permeants and are limited to specialized cell types. The transporters are low abundance proteins, and the equilibrative transporters of human and pig erythrocytes are the only ones that have been purified to homogeneity (4, 5). The equilibrative transporters have been subdivided on the basis of sensitivity to nitrobenzylthioinosine (NBMPR); one subtype (es) is inhibited by Յ1 nM NBMPR, as a result of high affinity (K d Ͻ 5 nM) binding of NBMPR at or near the permeant binding site (6 -8), whereas the other subtype (ei) is unaffected by low concentrations (Ͻ1 M) of NBMPR. The concentrative nucleoside transporters comprise several functional subtypes (1) that differ in their substrate selectivities and tissue distributions and, with some exceptions (9), are unaffected by high concentrations (Ͼ10 M) of NBMPR.Understanding of relationships among nucleoside transporters has been greatly advanced by the recent isolation and functional expression from rat and human cells of cDNAs encoding NT proteins reviewed in Ref. 3). These NT proteins comprise two structurally unrelated protein families that are designated ENT and CNT, depending on whether they mediate, respectively, equilibrative (E) or concentrative (C) NT processes. The mammalian ENT proteins identified thus far have approximately 450 amino acid residues (10, 11, 16) and 11 predicted transmembrane domains, whereas the mammalian CNT proteins have approximately 650 amino acid residues (12-14) and 14 predicted transmembrane domains. Members of the ENT family (10, 11, 16) exhibit functional characteristics that are typical of es-mediated (e.g. hENT1, rENT1) or ei-

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Section: Resultssupporting

confidence: 69%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Demonstration of Equilibrative Nucleoside Transporters (hENT1 and hENT2) in Nuclear Envelopes of Cultured Human Choriocarcinoma (BeWo) Cells by Functional Reconstitution in Proteoliposomes

Mani¹,

Hammond²,

Marjan³

et al. 1998

Journal of Biological Chemistry

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“…We (Vyas et al, 2002), endothelial cells (Hammond and Archer, 2004), and BeWo cells (Boumah et al, 1992).…”

Section: Discussionmentioning

confidence: 99%

Characterization of mENT1Δ11, a Novel Alternative Splice Variant of the Mouse Equilibrative Nucleoside Transporter 1

Robillard

Bone²,

Park³

et al. 2008

Mol Pharmacol

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Mammalian cells require specific transport mechanisms for the cellular uptake and release of endogenous nucleosides such as adenosine, and nucleoside analogs used in chemotherapy. We have identified a novel splice variant of the mouse equilibrative nucleoside transporter, mENT1, that results from the exclusion of exon 11 during pre-RNA processing. This variant encodes a truncated protein (mENT1⌬11) missing the last three transmembrane domains of the full-length mENT1. The mENT1⌬11 transcript and protein were found to be differentially distributed among tissues relative to full-length mENT1. PK15-NTD (nucleoside transport deficient) cells were transfected with mENT1 or mENT1⌬11 and assessed for nucleoside transport function. No significant differences were observed between the mENT1 and mENT1⌬11 in terms of transport function or inhibitor binding affinity. PK15-mENT1⌬11 transfected cells bound the ENT1 3 H]uridine. The only significant differences between the mENT1 variants were that mENT1⌬11 could not be photolabeled with [3 H]NBMPR and that mENT1⌬11 was insensitive to the transporter-modifying effects of N-ethylmaleimide. These data suggest that the last three transmembrane domains of mENT1 are not necessary for transport activity, but this region does contain the cysteines responsible for the sensitivity of mENT1 to sulfhydryl reagents, and the residues important for covalent modification of the protein with NBMPR. These results provide important guidelines for future mutagenesis studies aimed at elucidating the tertiary structure of the ENT1 protein and the domains involved in inhibitor binding and substrate translocation.

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Characterization of equilibrative and concentrative Na+-dependent (cif) nucleoside transport in acute promyelocytic leukemia NB4 cells

Roovers

Meckling‐Gill

1996

J. Cell. Physiol.

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Nucleoside transport processes can be classified by the transport mechanism, e=equilibrative and c=concentrative, by the sensitivity to inhibition by nitrobenzylthioinosine (NBMPR), s=sensitive and i=insensitive, and also by permeant selectivity. To characterize nucleoside transport in acute promyelocytic NB4 cells, nucleoside transport was resolved into different components by selective elimination of transport processes with NBMPR and with Na+-deficient media. Initial transport rates were estimated from time course experiments. For adenosine, uridine, and formycin B, equilibrative transport accounted for approximately 60% of their uptake, with ei and es transport contributing almost equally, and Na+-dependent transport accounting for the remaining 40% of the total uptake. Thymidine uptake was mediated exclusively by equilibrative systems with ei and es systems each contributing 50% to total uptake. Adenosine accumulated above equilibrative concentrations, suggesting that a concentrative transport process was active and/or that metabolism led to adenosine's accumulation. Formycin B, a nonmetabolizable analog, also accumulated in the cells, supporting the concentrative potential of the Na+-dependent transporter. Kinetic analyses also provided evidence for three distinct high affinity transport mechanisms. NBMPR binding assays indicated the presence of two high affinity (Km 0.10 and 0.35 nM) binding sites. In conclusion, NB4 cells express ei and es transport, as well as a large ci transport component, which appears to correspond to cif (f=formycin B or purine selective) nucleoside transport, not previously described in human cells.

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Expression of high levels of nitrobenzylthioinosine-sensitive nucleoside transport in cultured human choriocarcinoma (BeWo) cells

Cited by 44 publications

References 37 publications

Demonstration of Equilibrative Nucleoside Transporters (hENT1 and hENT2) in Nuclear Envelopes of Cultured Human Choriocarcinoma (BeWo) Cells by Functional Reconstitution in Proteoliposomes

Demonstration of Equilibrative Nucleoside Transporters (hENT1 and hENT2) in Nuclear Envelopes of Cultured Human Choriocarcinoma (BeWo) Cells by Functional Reconstitution in Proteoliposomes

Characterization of mENT1Δ11, a Novel Alternative Splice Variant of the Mouse Equilibrative Nucleoside Transporter 1

Characterization of equilibrative and concentrative Na+-dependent (cif) nucleoside transport in acute promyelocytic leukemia NB4 cells

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