Establishment of thiopurine S-methyltransferase gene knockdown in jurkat T-lymphocytes: an in vitro model of TPMT polymorphism.

Abstract

BACKGROUND:

Thiopurine S-methyltransferase (TPMT) is an excellent example of an enzyme whose pharmacogenetic polymorphisms affect efficacy and toxicity of a drug. The association between TPMT activity and thiopurine-related myelosuppression is well recognized. To study the significance of TPMT deficiency in thiopurine metabolism and immunosuppressive activity in vitro, we established RNA interference-based TPMT knockdown (kd) in a Jurkat cell line.

RESULTS:

In Jurkat TPMT kd cells, TPMT expression was reduced to 73% at the RNA level and 83% at the protein level. TPMT kd cells were more sensitive to 6-mercaptopurine (6-MP) (10 μmol/L) and 6-thioguanine (6-TG) (8 μmol/L) than wild-type (wt) cells, (32% versus 20%) and (18% versus 9%), respectively. Both Jurkat wt and kd cells were more sensitive to 6-TG-induced apoptosis than to 6-MP. 6-TG activity was also more affected by TPMT levels than was 6-MP as reflected by IC60, concentrations that is, 6-MP [4.6 μmol/L (wt) and 4.7 μmol/L (kd)], 6-TG [2.7 μmol/L (wt) and 0.8 μmol/L (kd)]. IC60 concentrations induced significant apoptosis in both Jurkat wt and kd cells (257%, versus 314%) with 6-MP and (323% versus 306%) with 6-TG, respectively. At IC60 (6-MP) 6-thioguanine nucleotides (6-TGN) accumulation in cells was 518 versus 447 pmol/million cells in wt and kd cells, respectively. On the other hand 6-TGN accumulation at IC60 (6-TG) was 477 versus 570 pmol/million cells in wt and kd cells, respectively. 6-Methylated mercaptopurine (6-MeMP) concentrations were more affected than 6-TGN by TPMT kd (194 versus 10 pmol/million cells) in wt and kd cells, respectively.

CONCLUSION:

We conclude that TPMT kd cells are an appropriate in vitro model to investigate the significance of TPMT deficiency with thiopurine therapy and could be helpful in understanding possible clinical consequences of TPMT polymorphism.

• Authors: Misbah Misdaq, Reiner Andag, Michael Oellerich, Abdul R Asif, Nicolas von Ahsen
• Journal: Therapeutic drug monitoring (Ther Drug Monit) Vol. 34 Issue 5 Pg. 584-92 (Oct 2012) ISSN: 1536-3694 [Electronic] United States
• PMID: 22972540 (Publication Type: Journal Article)

Chemical References

• Guanine Nucleotides
• Thionucleotides
• 6-thioguanylic acid
• Mercaptopurine
• Methyltransferases
• thiopurine methyltransferase
• Thioguanine

Topics

• Apoptosis (drug effects, genetics)
• Cell Line, Tumor
• Drug Hypersensitivity (enzymology, genetics)
• Gene Knockdown Techniques (methods)
• Guanine Nucleotides (genetics, metabolism)
• Humans
• Immune Tolerance
• Jurkat Cells
• Mercaptopurine (metabolism, pharmacology)
• Methyltransferases (deficiency, genetics)
• Polymorphism, Genetic (drug effects)
• Purine-Pyrimidine Metabolism, Inborn Errors (enzymology, genetics)
• T-Lymphocytes (drug effects, enzymology)
• Thioguanine (metabolism, pharmacology)
• Thionucleotides (genetics, metabolism)