Lentivirus-Mediated Expression of Mutant MGMT P140K Protects Human CD34 + Cells Against the Combined Toxicity of O 6 -Benzylguanine and 1,3-Bis(2-Chloroethyl)-Nitrosourea or Temozolomide

Abstract

Lentiviral vectors are capable of efficiently transducing nondividing and slowly dividing cells, including hematopoietic stem cells, resulting in stable integration and sustained transgene expression. We constructed human immunodeficiency virus type 1-based self-inactivating lentiviral vectors to express either wild-type or an O ⁶-benzylguanine (O ⁶-beG)-resistant mutant form of the human O ⁶-alkylguanine-DNA methyltransferase (MGMT; DNA-O ⁶-methylguanine:[protein]-L-cysteine S-methyltransferase, EC 2.1.1.63) and transduced K562 and granulocyte colony-stimulating factor-mobilized human peripheral blood CD34⁺ cells. After transduction, K562 cells expressed high levels of MGMT as determined by Western blot, immunocytochemistry, and biochemical assay. A colony-forming survival assay showed significant protection against O ⁶-beG plus 1,3-bis(2-chloroethyl)-nitrosourea (BCNU) or temozolomide (TMZ) toxicity. Similarly, a single transduction of CD34⁺ cells resulted in a 13- to 14-fold increase in the level of MGMT expression. In comparison with non-transduced cells, mutant MGMT^P140K-transduced CD34⁺ cells showed significant resistance against the combined toxicity of O ⁶-beG with either TMZ or BCNU: there was an ∼9-fold increase in the survival of colony-forming cells as indicated by the IC₅₀ values after O ⁶-beG plus TMZ treatment and an ∼5-fold increase in the case of O ⁶-beG plus BCNU treatment. These results show that lentivirus-mediated expression of MGMT^P140K can efficiently protect the hematopoietic compartment against the combined toxicity of O ⁶-beG plus TMZ or BCNU.