Human long-term culture initiating cells are sensitive to benzylguanine and 1,3-bis (2-chloroethyl)-1-nitrosourea and protected after mutant (G156A) methylguanine methyltransferase gene transfer

Human hematopoietic progenitors express low levels of O-6-alkylguanine-DNA alkyltransferase and are sensitive to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), particularly following O-6-benzylguanine (BG)-mediated O-6-alkylgua nine-DNA alkyltransferase inhibition. Expression of the BC-resistant mutant (C156A) methylguanine methyltransferase (Delta MGMT) gene in hematopoietic cells confers resistance to BG and BCNU. Because BCNU targets both early a nd late human hematopoietic cells and results in prolonged and cumulative m yelosuppression, we attempted to protect early hematopoietic progenitors (l ong-term culture initiating cells (LTC-ICs)) by retroviral-mediated transfe r of the Delta MGMT gene. A total of 33-56% of LTC-ICs were transduced with MFG-Delta MGMT retrovirus as determined by evidence of provirus in seconda ry colony-forming units at 5 weeks of culture under conditions optimal for the survival and proliferation of early hematopoietic progenitors. The addi tion of flt-3 ligand to cultures increased the transduction rate of LTC-ICs . Furthermore, 17.8 +/- 8.1% of Delta MGMT-transduced LTC-ICs survived dose s of BG and BCNU; these doses allowed the survival of only 0-1% of untransd uced LTC-ICs. This finding compares favorably with the 8-12% of CD34(+) cel l-derived colony-forming units that we previously showed became resistant t o BG and BCNU after Delta MGMT gene transfer. Thus, Delta MGMT transduction of human early hematopoietic progenitor LTC-ICs confers resistance to BC a nd BCNU and may allow transduced LTC-ICs selective survival and enrichment over untransduced cells in patients undergoing BC and BCNU chemotherapy.