Intracellular Superoxide Radical Formation Decreases 6‐Mercaptopurine Uptake by MOLT‐4 Leukemia Cells
Bibliographic record
Abstract
Background Acute lymphoblastic leukemia (ALL) is the most common cancer associated with childhood. 6‐Mercaptopurine (6‐MP) is a nucleobase analog drug used in the maintenance treatment phase of ALL. 6‐MP has numerous side‐effects associated with its use, which has both short‐term and long‐term implications which can impact patient compliance (especially in children). Our lab has established that SLC43A3, which encodes for the equilibrative nucleobase transporter 1 (ENBT1), is expressed in leukemia cells and transports 6‐MP. However, its modes of regulation are yet to be elucidated. A previous study in microvascular endothelial cells demonstrated that nucleobase uptake via ENBT1 was significantly decreased upon exposure to oxidative stress. Oxidative stress is very prevalent in cancer, including leukemia, suggesting that it could impact ENBT1 function and thus 6‐MP uptake and therapeutic activity. Hypothesis ENBT1 function will be reduced by oxidative stress and affect the cytotoxic efficacy of 6‐MP in leukemia cells. Methods The MOLT‐4 leukemia cell line was used for all experiments. Cell viability was assessed via the MTT assay. [ 14 C]6‐MP was used to determine the functional kinetics of ENBT1 using an oil‐stop centrifugation assay. Compounds used for the studies on oxidative stress were tert‐butyl hydroperoxide (TBHP) and menadione (oxygen‐free radical generators). TBHP causes extracellular induction of oxidative stress, while menadione is primarily through an intracellular mechanism called redox cycling. Mn(III)tetrakis(1‐methyl‐4‐pyridyl)porphyrin (MnTMPyP) (superoxide dismutase mimetic) and N‐acetylcysteine (NAC) (antioxidant) were used to combat the effects caused by TBHP or menadione. Results Incubation of cells in TBHP and menadione for 30 min at 37℃ decreased cell viability only at the highest concentrations tested (100 ‐ 400 µM) (400 µM: 86 ± 4% and 72 ± 6%, respectively) Incubation with TBHP and menadione for 24 hours led to a greater loss of cell viability (Log EC 50 : ‐3.9 ± 0.1 and ‐4.3 ± 0.1, respectively). TBHP did not alter ENBT1‐mediated 6‐MP uptake. However, incubation with menadione at 100 µM for 30 min at 37℃ significantly decreased ENBT1‐mediated 6‐MP uptake (V max : Control – 77 ± 3 pmol/µL/sec; Menadione – 57 ± 3 pmol/µL/sec). The addition of the free radical scavenger MnTMPyP did not reverse the decrease caused by menadione. However, the addition of 1mM NAC caused a significant reversal of the reduction in 6‐MP uptake produced by menadione (V max : Menadione – 46 ± 2 pmol/µL/sec; Menadione + NAC – 55 ± 2 pmol/µL/sec). Conclusions Menadione but not TBHP caused a significant decrease in ENBT1 functional activity, suggesting it was due to intracellular superoxide formation, thus supporting our hypothesis. The reduction caused by menadione could be reversed with NAC. This study confirms that ENBT1 function, and thus 6‐MP uptake by leukemia cells, is modified by oxidative stress, thus opening up potential therapeutic avenues in ALL treatment.
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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 | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.000 |
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".