The P140K mutant of human O6-methylguanine-DNA-methyltransferase (MGMT) confers resistance in vitro and in vivo to temozolomide in combination with the novel MGMT inactivator O6-(4-bromothenyl)guanine

✍ Scribed by Lorna B. Woolford; Thomas D. Southgate; Geoffrey P. Margison; Michael D. Milsom; Leslie J. Fairbairn

• Publisher: John Wiley and Sons
• Year: 2005
• Tongue: English
• Weight: 155 KB
• Volume: 8
• Category: Article
• ISSN: 1099-498X
• DOI: 10.1002/jgm.816

✦ Synopsis

The O 6 -methylguanine-DNA-methyltransferase (MGMT) inactivator O 6benzylguanine (O 6 -beG) is currently under clinical investigation as a potential tumour-sensitising agent. In clinical trials its use has been associated with increased myelotoxicity and a reduced maximum tolerated dose (MTD) for BCNU. Thus the concept of myeloprotection by gene therapy with an O 6 -beG-insensitive mutant of MGMT is soon to be tested. Recently, an alternative inactivator has been described (O 6 -(4-bromothenyl)guanine, PaTrin-2), which shows potential advantages over O 6 -beG in terms of higher activity against wild-type MGMT and oral formulation. The use of PaTrin-2 has also been associated with increased myelotoxicity in clinical trials and thus PaTrin-2 may also be a candidate for use in conjunction with mutant MGMT gene transfer in genetic chemoprotective strategies. However, its activity against mutant MGMTs has not been reported. We show here that the P 140 K mutant of MGMT is highly resistant to inactivation by PaTrin-2. Furthermore, we show that a human haemopoietic cell line (K562) transduced with a retroviral vector encoding MGMT P140K is highly resistant to the cytotoxic effects of PaTrin-2 in combination with the methylating agent temozolomide, and that cells expressing MGMT P140K can be effectively enriched in vitro following challenge with this drug combination. Finally, we show that animals reconstituted with bone marrow expressing MGMT P140K exhibit haemopoietic resistance to PaTrin-2/temozolomide, which results in in vivo selection of gene-modified cells. All of these effects were comparable to those also achieved using O 6 -beG/temozolomide. Thus our data show that MGMT P140K is a suitable candidate for chemoprotective gene therapy where PaTrin-2 is being used in conjunction with temozolomide.