Having shown that 6-MP promotes ATP depletion and the consequent activation of AMPK, we reasoned that metabolic modifications likely occur in response to this energetic stress. AICAR was used as a positive control.
The immunoblot is representative of four independent experiments. Bottom Histograms representing the densitometric analysis of the immunoblots. A schematic representation of genes implicated in glycolysis, glutaminolysis and nucleotide synthesis is shown at the right. Interestingly, this profile is similar albeit with higher intensity to that of induced by energetic stress e.
Even the physiological impact of these transcriptomic changes still requires further elucidation, it is conceivable that these expression profiles correspond to metabolic rewiring that would optimize glycolytic and glutaminolytic pathways e.
Together, our findings support that, by inhibiting ATP synthesis, 6-MP promotes energetic stress resulting into the altered expression of genes involved in nucleotide synthesis as well as glucose and glutamine metabolism. Having shown that 6-MP promotes energetic stress and impacts the expression of genes involved in glycolysis i.
These effects were maintained up to 72 h of exposition to 6-MP Supplementary Figure 4. Consistent with the downregulation of metabolic checkpoints in T cells, the metabolic activity of T cells was profoundly impacted by 6-MP.
Top Schematic representation of the procedure. The data are from three or five independent experiments. E Histograms representing the results of percentage of extracellular lactate as compared to vehicle. Cellular glucose influx is a rate-limiting step of glycolysis, and we observed global shutdown of glucose metabolism in cells exposed to 6-MP.
Therefore, we asked whether 6-MP can reduce glucose uptake to cause energetic stress and metabolic disruption. Together, these results indicate that the metabolic alterations associated with 6-MP are likely not caused by inhibition of cellular glucose uptake. A Left Schematic representation of the procedure.
The immunoblot is representative of three independent experiments. We provide for the first time a comprehensive characterization of the impact of 6-MP exposition in proliferating T cell metabolism. Our results indicate that 6-MP produces an early drop in ATP production followed by transcriptional reprogramming of genes involved in glycolysis, glutaminolysis and nucleotide synthesis; these changes are similar to those observed upon energetic stress. Besides the inhibition of purine synthesis, the biochemical basis for rapid ATP depletion induced by 6-MP remains to be established.
In addition, ATP depletion induced by 6-MP could impact glycolytic and glutaminoytic fluxes as a result of the inhibition of ATP-dependent enzymes, such as hexokinase. These results provide new insights into how incubation with 6-MP, which induces ATP depletion, promotes metabolic stress and adds a possible mechanism by which 6-MP inhibits cell proliferation. In addition, this finding underscores the critical role of ATP production in proliferating T cells, including the Jurkat T cell line [ 38 ].
Consistent with this, we have recently demonstrated that 6-MP-resistant lymphoblastoid cell lines grow more slowly than 6-MP-sensitive cells, suggesting the importance of basal proliferation rate of cells in anticipating the antiproliferative action of 6-MP [ 15 ]. Together, these results underscore the critical role of the maintain of ATP homeostasis in leukemic T cells and offer the opportunity to reassess the beneficial effects of 6-MP as a therapeutic approach in association with other antiproliferative agents that would target multiple metabolic pathways crucial for cell proliferation.
In addition, it would be interesting to determine if variations in key molecules regulating glycolysis and glutaminolysis could be surrogate biomarkers of the interindividual variability in the clinical response to 6-MP by and if so monitoring metabolic reprogramming could be useful to predict 6-MP efficacy. Whether the effect of 6-MP on the metabolic reprogramming of Jurkat T cells can be generalize to other situations during which T cells rapidly grow such as naive T cells upon antigen presentation that is, in a non-oncological setting remains to be demonstrated.
Our results cannot be directly translated into medical situation other that leukemia because Jurkat cells may not be informative of normal T cell responses. This cell line is not dependent on T-cell receptor TCR signals for nutrient utilization, cell cycle progression and growth. Hence, our findings are relevant for leukemia-related situations, but cannot be directly extrapolated into adaptive immunity. Along these lines, deciphering the impact of other immunosuppressive agents on metabolic reprogramming, including mycophenolate mofetil which inhibits the rate-limiting enzyme of de novo purine synthesis, could yield new insights on its mechanisms of action with important therapeutic implications.
Indeed, identifying molecular circuitries that regulate cell metabolism and could eventually be targeted by immunosuppressive drugs is a strong approach to design drugs that block metabolic checkpoints in a complementary manner. Nevertheless, the impact of 6-MP on metabolic reprogramming appears to be cell-dependent, as opposing effects have been reported. Regarding glucose uptake, in the L6 model of rat skeletal muscle, 6-MP increases glucose uptake and Glut4 SLC2A4, solute carrier family 2, member 4 translocation to the cell surface, a process mediated by NR4A3 [ 42 ].
The fact that T cells do not express Glut4 [ 37 ] may explain why 6-MP does not promote increased glucose uptake in our model.
Since the precise mechanisms that cause the adverse effects of 6-MP e. In addition, multiple mechanisms are likely involved in the regulation of critical metabolic pathways by 6-MP, and which may be cell-type dependent.
Nevertheless, these distinct mechanisms are not exclusive, and whether P53 signaling is activated upon 6-MP exposure remains to be determined. The impact of the energetic stress induced by 6-MP on cell viability, and in comparison with established mechanisms such as incorporation 6-TG in DNA, Rac-1 inhibition and de novo purine synthesis remains to be established.
However, the specific delineation of the impact of the energetic stress induced by 6-MP is complicated by the interdependence of the signaling pathways activated in the same time by 6-MP. For example, DNA damage induced by 6-TG activate P53 signaling, which, in turn, can impact metabolic checkpoints and cell metabolism.
In addition, the metabolic changes induced by 6-MP correspond to an adaptive process, and AMPK activation, mTOR inhibition and autophagy have prosurvival, rather than proapoptotic properties. In conclusion, our findings offer new insights on the cellular effects of 6-MP on metabolic alterations by demonstrating that 6-MP promotes an early energetic stress that impacts proliferation and increases apoptosis in proliferating T cells.
Finally, our findings might provide an original rational approach to better redesign therapeutic combinations of antiproliferative agents with the aim of controlling cell metabolism by targeting different metabolic pathways.
Jurkat cells are T lymphocytes obtained from a peripheral blood of 14 years-old male with acute T cell leukemia, and they express CD3 and CD The cell apoptosis assay was performed as described previously [ 44 ]. Cell morphology was studied using a fluorescence microscope. Between and cells were counted per condition.
Live cells were stained green, and apoptotic cells were stained orange with shrunken and fragmented nuclei. Among all cells counted, the percentage of apoptotic cells was calculated.
Comparison of the expression of genes after exposition was visualized as heat maps. Primers sequences are listed in Supplementary Table 1. Immunoblotting was performed as previously described [ 47 ]. Luminescence was measured at a microplate luminescence counter Enspire Multilabel Reader Perkin Elmer. Each condition was measured in triplicate.
Luminescence was measured as for ATP detection assay. The absorption values were plotted against a lactate standard curve to determine the lactate concentrations in the samples. Glucose and glutamine oxidation flux were determined by the rate of 14 CO 2 released from 14 C-U-glucose and 14 C-U-glutamine, respectively. Half of the sample content was transferred into scintillation vials, and the radiolabelled glucose incorporated into the cells was measured by using Ultima Gold and reading the samples on a liquid scintillation counter.
The data were analysed with FlowJo software TreeStar. The distribution of variables is represented using box-and-whisker plots. The distributions are represented using histograms. We used the Mann-Whitney U test for nonparametric data comparisons between two groups and a t -test to compare the parametric data. Statistical analysis were performed using GraphPad Prism software version 5. Mercaptopurine is in a class of medications called purine antagonists.
It works by stopping the growth of cancer cells. Mercaptopurine comes as a tablet and a suspension liquid to take by mouth. It is usually taken once a day. Take mercaptopurine at around the same time every day. Follow the directions on your prescription label carefully, and ask your doctor or pharmacist to explain any part you do not understand. Take mercaptopurine exactly as directed. Do not take more or less of it or take it more often than prescribed by your doctor.
If you are taking the suspension, shake the bottle very well for 30 seconds before each use to mix the medication evenly. It is important to use an oral syringe measuring device to accurately measure and take your dose of mercaptopurine.
If you do not find an oral syringe with your medication, ask your pharmacist to give you one. After you use the oral syringe to take your medication, remove the plunger from the rest of the measuring device, wash both parts with warm soapy water, and rinse under running tap water.
Allow the parts to air dry before putting back together for the next use. Continue to take mercaptopurine even if you feel well. Do not stop taking mercaptopurine without talking to your doctor. Mercaptopurine is also sometimes used to treat certain other types of cancer, Crohn's disease a condition in which the body attacks the lining of the digestive tract causing pain, diarrhea, weight loss, and fever , and ulcerative colitis condition in which sores develop in the intestines causing pain and diarrhea.
Talk to your doctor about the possible risks of using this medication for your condition. This medication may be prescribed for other uses; ask your doctor or pharmacist for more information. Take the missed dose as soon as you remember it. However, if it is almost time for the next dose, skip the missed dose and continue your regular dosing schedule.
Do not take a double dose to make up for a missed one. Taking mercaptopurine may increase the risk that you will develop a new cancer. Some people who took mercaptopurine to treat Crohn's disease or ulcerative colitis developed hepatosplenic T cell lymphoma HSTCL , a very serious form of cancer that often causes death within a short time. Tell your doctor if you experience any of the following symptoms: stomach pain; fever; unexplained weight loss; night sweats or easy bruising or bleeding.
Talk to your doctor about the risks of taking this medication. Chemotherapy drugs that affect cells only when they are dividing are called cell-cycle specific. Chemotherapy drugs that affect cells when they are at rest are called cell-cycle non-specific. The scheduling of chemotherapy is set based on the type of cells, rate at which they divide, and the time at which a given drug is likely to be effective.
This is why chemotherapy is typically given in cycles. Chemotherapy is most effective at killing cells that are rapidly dividing. Unfortunately, chemotherapy does not know the difference between the cancerous cells and the normal cells. The "normal" cells will grow back and be healthy but in the meantime, side effects occur.
Different drugs may affect different parts of the body. Mercaptopurine belongs to the class of chemotherapy drugs called antimetabolites. Antimetabolites are very similar to normal substances within the cell. When the cells incorporate these substances into the cellular metabolism, they are unable to divide.
Antimetabolites are cell-cycle specific. They attack cells at very specific phases in the cycle. Antimetabolites are classified according to the substances with which they interfere.
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