Summary Mechanisms of resistance to the active metabolite 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MTIC) of the drug 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC) were studied in three human cell lines with differing amounts of the repair enzyme 06-alkylguanine-DNA alkyltransferase (06AT). The lines were HT29 (Mer+Rem+), A549 (Mer+Rem-) and VA13 (Mer-). The ability to repair 06 methylguanine was directly related to resistance to MTIC (HT29 ID50 650 pmol -1, A549 ID50 210 pmol -1, VA1 3 ID50 15 pmol -1). MTIC produced DNA single strand breaks over the range of one log of cell kill, but depletion of cellular NAD levels could not be detected until there was greater than 95% cell kill. Inhibitors of the repair enzyme adenosine diphosphoribosyl transferase (ADPRT) potentiated killing by 2-fold in the Mer+ cell lines but not the Mer-line. The enhancement was directly proportional to an increase in DNA strand breaks but not a change in their half-life. Therefore resistance to the clinically used methylating agent MTIC can be partly overcome by inhibiting ADPRT but a role for ADPRT as a suicide mechanism in response to alkylating agent damage is unlikely.
Histone preparationChromatin was isolated from the livers of adult rats (Scott-Russ strain) according to the method of Bonner et al. (1968), omitting centrifugation through dense sucrose solution. Histone was extracted from the isolated chromatin by blending with 0-25N HCI. The insoluble material was centrifuged off and the extraction procedure repeated. The supernatants were pooled and histone was precipitated from the acid extract by adding 9 volumes of acetone. The precipitate was washed with acetone, air dried and finally vacuum dried.
Tissue culture method8The technique adopted to study invasion in vitro involved the use of modified Trowell organ culture flasks. The apparatus (Fig. 1)
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