Use of antibodies to human O⁶-alkylguanine-DNA alkyltransferase to study the content of this protein in cells treated with O⁶-benzylguanine or N-methyl-N′-nitro-N-nitrosoguanidine

Abstract

Antisera raised in rabbits to three peptides corresponding to amino acid sequences found in human O6-alkylguanine-DNA alkyltransferase were used to study the fate of the alkyltransferase protein in human colon tumor cells after exposure to N-methyl-N'-nitro-N-nitrosoguanidine or to O6-benzylguanidine. Under these conditions, the alkyltransferase protein becomes inactivated, presumably by the conversion of its cysteine acceptor site to S-methylcysteine or S-benzylcysteine respectively. It was found that the protein was rapidly degraded after such inactivation both in intact cells and in cell-free extracts. It is probable that a conformational change in the protein is brought about by conversion of the alkyltransferase to the inactive form by alkylation of the cysteine acceptor site. This change may render the protein very sensitive to proteolytic degradation. The rapid degradation of the inactive form of the protein may serve as a signal for its resynthesis but in the short term ensures that its reactivation by regeneration of the cysteine acceptor site is unlikely to occur to any significant extent. The short half-life of the inactivated alkyltransferase protein makes it probable that measurement of the content of the alkyltransferase protein by immunohistochemistry, which is likely to measure the sum of the active and inactivated forms of the protein, will nevertheless yield an accurate estimation of the cellular capacity to repair O6-methylguanine provided that procedures with sufficient specificity and affinity can be developed.

Expression of the inactive C145A mutant human O6-alkylguanine-DNA alkyltransferase in E.coli increases cell killing and mutations by N-methyl-N′-nitro-N-nitrosoguanidine

“…The fate of the methylated form of the AGT protein that results from repair of DNA is not fully understood but several studies have shown that the protein undergoes a conformational change after alkylation of the active site cysteine (56-58) and is degraded rapidly (59,60). There is also evidence that the alkylated form of the protein becomes ubiquitinated (60,61).…”

A Novel Temozolomide–Perillyl Alcohol Conjugate Exhibits Superior Activity against Breast Cancer Cells In Vitro and Intracranial Triple-Negative Tumor Growth In Vivo

“…This feature is exploited by the use of specific MGMT inhibitors, such as O6-benzylguanine (O6-BG), which act as pseudosubstrates (15). Benzylation of MGMT via reaction with O6-BG causes the same structural change in the enzyme as that seen after alkylation following DNA repair, and therefore also leads to rapid degradation of the protein (16). Ablation of MGMT activity after treatment of MGMT-positive cells with O6-BG generally increases their sensitivity to killing by TMZ, and this has been well established in numerous in vitro and in vivo tumor models (see detailed in refs.…”

Point mutations at multiple sites including highly conserved amino acids maintain activity, but render O6-alkylguanine‒DNA alkyltransferase insensitive to O6-benzylguanine

“…The Salkylcysteine formed at the active site of AGT is not converted back into cysteine ; the protein, therefore, can act only once. The alkylated form of the AGT protein undergoes a conformational change, which leads to its rapid degradation, possibly by facilitating its ubiquitinylation [6][7][8].…”