EXTENDED DEPLETION OF O-6-METHYLGUANINE-DNA METHYLTRANSFERASE ACTIVITY FOLLOWING O-6-BENZYL-2'-DEOXYGUANOSINE OR O-6-BENZYLGUANINE COMBINEDWITH STREPTOZOTOCIN TREATMENT ENHANCES 1,3-BIS(2-CHLOROETHYL)-1-NITROSOUREA CYTOTOXICITY

We have recently suggested that optimal reversal of 1,3-bis(2-chloroet hyl)-1-nitrosourea (BCNU) resistance might require complete inactivati on of the DNA repair protein O-6-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 r egeneration rate of MGMT activity following O-6-benzylguanine (BG), O- 6-benzyl-2'-deoxyguanosine (dBG), and streptozotocin (STZ) in determin ing the potentiation of BCNU cytotoxicity in the highly resistant colo n carcinoma cell line HT-29. To this end, we measured the enhancement of BCNU cytotoxicity utilizing regimens which provided complete inhibi tion, with partial or complete recovery of MGMT activity by 24 h. We w ere able to modulate the recovery rate of MGMT activity following BG o r dBG administration by repeated washing of cells with complete medium . Subsequent to equally inhibitory doses of BG (100 mu M) or dBG (1.0 mM) treatment without mashing, MGMT activity was completely inactivate d 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 comple te medium. This recovery was completely inhibited for 24 h by combinin g BG or dBG with 2.5 mM STZ. These differential repletion profiles pro duced 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) potentiat ed BCNU cytotoxicity by approximately 3 logs of synergistic cell kill. When the recovery rate of MGMT activity was markedly enhanced via was hing of cells, BG-BCNU or dBG-BCNU produced less than 1 log of synergi stic cell kill. The addition of STZ to BG or dBG inhibited this tempor al recovery for 24 h and potentiated BCNU cytotoxicity by approximatel y 4 logs. These data further demonstrate that extended depletion of MG MT is required for optimal reversal of BCNU resistance. Because a thre e-drug combination of BG-STZ BCNU or dBG-STZ-BCNU consistently produce d greater cytotoxicity than any two-drug regimen, clinical testing of these combinations is warranted. Additionally, our data suggest that t he design of clinical regimens targeting the inactivation of MGMT and the reversal of BCNU resistance should consider the functional importa nce of extended depletion of MGMT in order to increase the possibility of antitumor responses.