Lentivirus-mediated expression of mutant MGMTP140K protects human CD34+ cells against the combined toxicity of O6-benzylguanine and 1,3-bis(2-chloroethyl)-nitrosourea or temozolomide.
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 O6-benzylguanine (O6-beG)-resistant mutant form of the human O6-alkylguanine-DNA methyltransferase (MGMT; DNA-O6-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 O6-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 MGMTP140K-transduced CD34+ cells showed significant resistance against the combined toxicity of O6-beG with either TMZ or BCNU: there was an approximately 9-fold increase in the survival of colony-forming cells as indicated by the IC50 values after O6-beG plus TMZ treatment and an approximately 5-fold increase in the case of O6-beG plus BCNU treatment. These results show that lentivirus-mediated expression of MGMTP140K can efficiently protect the hematopoietic compartment against the combined toxicity of O6-beG plus TMZ or BCNU.
Vince Lombardi Gene Therapy Laboratory, Immunotherapy Program, St. Luke's Medical Center, Milwaukee, WI 53151, USA.
Dhanalakshmi
D
Chinnasamy
Leslie J
LJ
Fairbairn
James
J
Neuenfeldt
Jonathan S
JS
Treisman
John P
JP
Hanson
Geoffrey P
GP
Margison
Nachimuthu
N
Chinnasamy
D018952:Antigens, CD34 / Q000378:metabolism; D015153:Blotting, Western; D002330:Carmustine / Q000633:toxicity; D002460:Cell Line; D003114:Colony-Forming Units Assay; D017931:DNA Primers; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000633:toxicity; D005434:Flow Cytometry; D015971:Gene Expression Regulation, Enzymologic; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D007150:Immunohistochemistry; D020128:Inhibitory Concentration 50; D016086:Lentivirus / Q000235:genetics*; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics*; D016133:Polymerase Chain Reaction; D000077204:Temozolomide; D014161:Transduction, Genetic; D019076:Transgenes / Q000235:genetics; D014407:Tumor Cells, Cultured
D018952:Antigens, CD34; D017931:DNA Primers; D003606:Dacarbazine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D002330:Carmustine; D000077204:Temozolomide