title: Quantitative relationship between guanine O(6)-alkyl lesions produced by Onrigin™ and tumor resistance by O(6)-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.
authors.affiliation: Department of Pharmacology, Cancer Center, Yale University School of Medicine, New Haven, CT 06520, United States.
authors.forename: Kimiko
authors.initials: K
authors.lastname: Ishiguro
authors.forename: Yong-Lian
authors.initials: YL
authors.lastname: Zhu
authors.forename: Krishnamurthy
authors.initials: K
authors.lastname: Shyam
authors.forename: Philip G
authors.initials: PG
authors.lastname: Penketh
authors.forename: Raymond P
authors.initials: RP
authors.lastname: Baumann
authors.forename: Alan C
authors.initials: AC
authors.lastname: Sartorelli
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002330:Carmustine / Q000494:pharmacology; D045744:Cell Line, Tumor; D004249:DNA Damage; D019175:DNA Methylation; D004260:DNA Repair; D003606:Dacarbazine / Q000031:analogs & derivatives / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis; D006801:Humans; D006834:Hydrazines / Q000494:pharmacology*; D051379:Mice; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000032:analysis / Q000502:physiology*; D013449:Sulfonamides / Q000494:pharmacology*; D000077204:Temozolomide
chemical_list: D000970:Antineoplastic Agents; D006834:Hydrazines; D013449:Sulfonamides; C483604:laromustine; D006147:Guanine; D003606:Dacarbazine; C008449:O-(6)-methylguanine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D002330:Carmustine; D000077204:Temozolomide