Quantitative relationship between guanine O6-alkyl lesions produced by Onrigin™ and tumor resistance by O6-alkylguanine-DNA alkyltransferase

Abstract

O 6 -Alkylguanine-DNA alkyltransferase (AGT) mediates tumor resistance to alkylating agents that generate guanine O 6 -chloroethyl (Onrigin ™ and carmustine) and O 6 -methyl (temozolomide) lesions; however, the relative efficiency of AGT protection against these lesions and the degree of resistance to these agents that a given number of AGT molecules produces are unclear. Measured from differential cytotoxicity in AGT-ablated and AGT-intact HL-60 cells containing 17,000 AGT molecules/cell, AGT produced 12-and 24-fold resistance to chloroethylating (90CE) and methylating (KS90) analogs of Onrigin ™ , respectively. For 50% growth inhibition, KS90 and 90CE generated 5,600 O 6 -methylguanines/cell and ~300 O 6 -chloroethylguanines/cell, respectively. AGT repaired O 6 -methylguanines until the AGT pool was exhausted, while its repair of O 6 -chloroethylguanines was incomplete due to progression of the lesions to AGT-irreparable interstrand DNA cross-links. Thus, the smaller number of O 6 -chloroethylguanine lesions needed for cytotoxicity accounted for the marked degree of resistance (12-fold) to 90CE produced by AGT. Transfection of human or murine AGT into AGT deficient transplantable tumor cells (i.e., EMT6, M109 and U251) generated transfectants expressing AGT ranging from 4,000 to 700,000 molecules/cell. In vitro growth inhibition assays using these transfectants treated with 90CE revealed that AGT caused a concentration dependent resistance up to a level of ~10,000 AGT molecules/cell. This finding was corroborated by in vivo studies where expression of 4,000 and 10,000 murine AGT molecules/cell rendered EMT6 tumors partially and completely resistant to Onrigin ™ , respectively. These studies imply that the antitumor activity of Onrigin ™ stems from guanine O 6 -chloroethylation and define the threshold concentration of AGT that negates its antineoplastic activity.

Distinct mechanisms of cell-kill by triapine and its terminally dimethylated derivative Dp44mT due to a loss or gain of activity of their copper(II) complexes

“…Growth inhibition assays based on cell counts using HL-60 cells continuously exposed to various agents for 3 days were previously described [29]. Clonogenic survival assays using MCF-7 cells were carried out as described [30].…”

Expression of O<sup>6</sup>-Methylguanine-DNA Methyltransferase Examined by Alkyl-Transfer Assays, Methylation-Specific PCR and Western Blots in Tumors and Matched Normal Tissue

“…These two types of alkylating agents exert cytotoxicity through distinctive mechanisms, chloroethylating agents via the generation of highly lethal interstrand DNA cross-links and methylating agents via an intact mismatch repair system [3]. Although MGMT produces marked tumor resistance to both types of alkylating agents, the underlying mechanisms are different [6]. MGMT repairs the methyl lesions with enormous efficiency until the MGMT pool is exhausted.…”

Preliminary Studies with a New Hypoxia-Selective Cytotoxin, KS119W,In VitroandIn Vivo

“…4). AGT transfers guanine O 6 -alkyl groups to the cysteine in the active site of AGT and thereby restores the O-6 position of the guanine to its native state, preventing the formation of crosslinks (11, 29–24, 26, 30). EMT6 cells transfected with the gene for either human or mouse AGT, and expressing either 10,000 molecules/cell of mouse AGT or 18,000 molecules/cell of human AGT, respectively, showed markedly increased resistance to the cytotoxic effects of KS119W in hypoxia (Fig.…”

“…Two features of EMT6 cells are important in these studies. First, they lack detectable O 6 -alkylguanine-DNA alkyltransferase (AGT), which is also known as O 6 -methylguanine DNA-methyltransferase (MGMT) (23, 26, 30). Second, they can be grown as solid tumors in mice, as well as in cell culture (27, 28, 31–32).…”

“…EMT6 cells were maintained in Waymouth’s Medium with 15% fetal bovine serum and antibiotics. EMT6 cells transfected with murine and human AGT cDNAs were developed and characterized by Ishiguro (30) and Baumann (23), respectively, and were cultured in McCoy’s 5A medium with 10% fetal calf serum and antibiotics. When being compared with these transfected cells, parental EMT6 cells were also grown in McCoy’s 5A medium.…”