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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.

journal_name

Ther Drug Monit

journal_title

Therapeutic drug monitoring

authors

Misdaq M,Andag R,Oellerich M,Asif AR,von Ahsen N

doi

10.1097/FTD.0b013e31826ec4b4

subject

Has Abstract

pub_date

2012-10-01 00:00:00

pages

584-92

issue

eissn

0163-4356

issn

1536-3694

pii

00007691-201210000-00016

journal_volume

pub_type

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