Extended depletion of O6-methylguanine-DNA methyltransferase activity following O6-benzyl-2'-deoxyguanosine or O6-benzylguanine combined with streptozotocin treatment enhances 1,3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity

We have recently suggested that optimal reversal of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) resistance might require complete inactivation of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) for at least 24 h following BCNU administration (22). This study was undertaken to further evaluate the functional importance of the regeneration rate of MGMT activity following O6-benzylguanine (BG), O6-benzyl-2'-deoxyguanosine (dBG), and streptozotocin (STZ) in determining the potentiation of BCNU cytotoxicity in the highly resistant colon carcinoma cell line HT-29. To this end, we measured the enhancement of BCNU cytotoxicity utilizing regimens which provided complete inhibition, with partial or complete recovery of MGMT activity by 24 h. We were able to modulate the recovery rate of MGMT activity following BG or dBG administration by repeated washing of cells with complete medium. Subsequent to equally inhibitory doses of BG (100 microM) or dBG (1.0 mM) treatment without washing, MGMT activity was completely inactivated for 24 h. However, MGMT activity recovered to control levels by 24 h when cells were treated with BG or dBG and washed 4 times with complete medium. This recovery was completely inhibited for 24 h by combining BG or dBG with 2.5 mM STZ. These differential repletion profiles produced disparate potentiation of BCNU cytotoxicity. The regimens which produced complete inactivation of MGMT for 24 h produced the greatest enhancement of BCNU cytotoxicity. BG or dBG (without a wash) potentiated BCNU cytotoxicity by approximately 3 logs of synergistic cell kill. When the recovery rate of MGMT activity was markedly enhanced via washing of cells, BG-BCNU or dBG-BCNU produced less than 1 log of synergistic cell kill. The addition of STZ to BG or dBG inhibited this temporal recovery for 24 h and potentiated BCNU cytotoxicity by approximately 4 logs. These data further demonstrate that extended depletion of MGMT is required for optimal reversal of BCNU resistance. Because a three-drug combination of BG-STZ-BCNU or dBG-STZ-BCNU consistently produced greater cytotoxicity than any two-drug regimen, clinical testing of these combinations is warranted. Additionally, our data suggest that the design of clinical regimens targeting the inactivation of MGMT and the reversal of BCNU resistance should consider the functional importance of extended depletion of MGMT in order to increase the possibility of antitumor responses.