Development of a Novel HEK293 Cell Model Lacking SLC29A1 to Study the Pharmacology of Endogenous SLC29A2-Encoded Equilibrative Nucleoside Transporter Subtype 2
Bibliographic record
Abstract
Equilibrative nucleoside transporters (ENT) mediate the transmembrane flux of endogenous nucleosides and nucleoside analogues used clinically. The predominant subtype, ENT1, has been well characterized. However, the other subtype, ENT2, has been less well characterized in its native milieu due to its relatively low expression and the confounding influence of co-expressed ENT1. We created a cell model where ENT1 was removed from HEK293 cells using CRISPR/cas9 (ENT1KO cells); this cell line has ENT2 as the only functional purine transporter. Transporter function was assessed through measurement of [3H]2-chloroadenosine uptake. ENT1 protein was quantified based on the binding of [3H]nitrobenzylthioinosine, and ENT1/ENT2 protein was detected by immunoblotting. Changes in expression of relevant transporters and enzymes involved in purine metabolism were examined by qPCR. Wildtype HEK293 cells and ENT1KO cells had a similar expression of SLC29A2/ENT2 transcript/protein and ENT2-mediated [3H]2-chloroadenosine transport activity (Vmax values of 1.02 {plus minus} 0.06 and 1.50 {plus minus} 0.22 pmol/µl/s, respectively). Of the endogenous nucleosides/nucleobases tested, adenosine had the highest affinity (Ki) for ENT2 (2.6 µM), while hypoxanthine was the only nucleobase with a sub-millimolar affinity (320 µM). A range of nucleoside/nucleobase analogues were also tested for their affinity for ENT2 in this model, with affinities (Ki) ranging from 8.6 µM for ticagrelor to 2,300 µM for 6-mercaptopurine. Our data suggest that the removal of endogenous ENT1 from these cells does not change the expression or function of ENT2. This cell line should prove useful for the analysis of novel drugs acting via ENT2 and to study ENT2 regulation. Significance Statement We have created a cell line whereby endogenous ENT2 can be studied in detail in the absence of the confounding influence of ENT1. Loss of ENT1 has no impact on the expression and function of ENT2. This novel cell line will provide an ideal model for studying drug interactions with ENT2 as well as the cellular regulation of ENT2 expression and function.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.002 |
| Insufficient payload (model declined to judge) | 0.002 | 0.002 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".