RNA Interference Targeting Thiopurine Methyl Transferase Gene Alters the Cytotoxic Effect of the 6-Mercaptopurine In Human Leukaemia Cells

Abstract 2891

Thiopurines; mercaptopurine (6-MP) and 6-thioguanine (6-TG) are important drugs in treatment of paediatric cancer patients. The activity of these drugs depends on the activity of several common enzymes in the metabolism pathways such as thiopurine methyl transferase (TPMT) and guanine monphosphate synthetase (GMPS). In the present study the efficacy of thiopurines was investigated upon inhibition of TPMT and GMPS gene expression by RNA interference (siRNA). Treatment of the MOLT4 human T-cell leukemia cells with TPMT and GMPS siRNA resulted in decreased mRNA expression as determined by Real-Time PCR by 60% and 70% respectively. When reducing TPMT mRNA, the MOLT-4 cells were 70% less sensitive to 6-MP while the sensitivity to 6-TG was unchanged. When down-regulating GMPS using siRNA the sensitivity was unchanged upon treatment with both drugs. A microarray experiment was conducted using wild type on MOLT-4, 6-MP and 6-TG resistant MOLT-4 variants. The aim was to identify affected genes in response to the resistance induction. The mRNA levels of several nucleoside transporter genes were down-regulated in both thiopurine-resistant sub-lines; concentrative nucleoside transporter 3 (CNT3) and equilibrative nucleoside transporter 2 (ENT2). The expression of genes encoding the purine de novo synthesis enzymes was reduced to the same extent in both resistant cell lines as well as the expression of GMPS (>40% in both resistant sub-lines) which indicates defected purine metabolism. The activity of TPMT and other enzymes in metabolism pathway of 6-MP and 6-TG was unchanged in resistant cells. Our results suggest that the contribution of TPMT activity in cytotoxicity of 6-MP is greater than for 6-TG, probably due to inhibition of de novo purine synthesis by methylated nucleotides, whereas TPMT inactivates 6-TG by methylation.