Establishment of human lymphoma cell lines with different thiopurine S-methyltransferase (TPMT) activities and differential proteome analysis after thiopurine exposure.
by Misbah Misdaq
Date of Examination:2012-12-12
Date of issue:2013-04-09
Advisor:Prof. Dr. A. R. Rahman
Referee:PD Dr. Wilfried Kramer
Referee:Prof. Dr. Jürgen Brockmöller
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Name:Misbah Misdaq dissertation 270313.pdf
EnglishThiopurines are purine analogues used as immunosuppressive and antileukemic agents. Thiopurine activation requires extensive metabolism that involves many enzymatic reactions and TPMT is one of the crucial enzymes in the thiopurine metabolic pathway. The present study aimed to establish a TPMT stable knockdown model to serve as an in vitro model of TPMT deficiency, its characterization, and its use to understand the importance of TPMT SNPs in thiopurine therapy as well as the possible further exploitation of model for the identification of novel thiopurine cellular targets. Jurkat T-lymphocytes were transfected with pSi.2.1 containing TPMT specific shRNA. A clone with maximum TPMT knockdown (kd) was selected and compared with and without exposure to thiopurines to non-transfected control cells (wt). Growth inhibition studies on wt and kd cells using 6-MP and 6-TG showed differential effects of TPMT expression. TPMT kd cells were many fold more sensitive to 6-TG compared to wt cells as measured by their IC60 doses. Similarly, TPMT deficient cells are more sensitive to 6-TG induced apoptosis. TPMT kd cells had less methylation capacity for 6-TG and hence accumulated more active metabolite (i.e. 6-TGN) and as a result a higher apoptosis rate. We further investigated novel cellular targets of thiopurine therapy using cells with different levels of TPMT expression. We utilized a 2-DE mass spectrometric approach to examine effects on wt and kd cells treated with IC60 doses of 6-MP and 6-TG. A total of thirteen proteins showed altered expression and nine proteins showed altered phosphorylation in all groups. Identified altered proteins fall into different functional categories; performing crucial roles in cellular homeostasis, cell cycle regulation, DNA damage response, apoptosis, cytoskeleton dynamics, energy metabolism and oxidative stress response. Among the identified altered proteins, four proteins (i.e. THIO, TXD17, GSTM3 and PRDX3) are known to be involved in the oxidative stress response of cells. This observation was further confirmed by ROS assay. Significant increase in oxidative stress was observed in wt and kd cells exposed to thiopurines confirming the proteome findings. Hence, we report for the first time the regulation of important oxidative stress related proteins by TPMT and thiopurines. This TPMT knockdown model demonstrates all the characteristics of human TPMT deficiency that can serve as an in vitro model of TPMT polymorphism.