Ubiquitination-Dependent Proteolysis of O⁶-Methylguanine-DNA Methyltransferase in Human and Murine Tumor Cells following Inactivation with O⁶-Benzylguanine or 1,3-Bis(2-chloroethyl)-1-nitrosourea

Abstract

In this study, we investigated the role of ubiquitination in the disposition of the inactivated O6-methylguanine-DNA methyltransferase (MGMT) protein in human (HT-29 and CEM) and murine (ts85) tumor cells. Using a combination of immunoprecipitation and immunoblotting techniques with antibodies against ubiquitin and MGMT, and anti-ubiquitin immunoaffinity chromatography, the MGMT protein was found to coexist with small amounts of its ubiquitinated species in both human and mouse tumor cells, suggesting the presence of endogenous inactivated MGMT. Further, treatment of HT-29 and CEM cells with MGMT-inactivating compounds, O6-benzylguanine (O6-BG, 20 microM) or 1,3-bis(chloroethyl)-1-nitrosourea (BCNU, 100 microM), resulted in increased levels of ubiquitinated MGMT within 1.5-3 h of drug exposure. Kinetic studies in HT-29 cells treated with O6-BG indicated a slow and gradual conversion of the inactivated MGMT to its polyubiquitinated forms over a course of 3-18 h, with a concomitant disappearance of the parent MGMT protein. We also characterized the previously reported O6-BG-induced degradation of MGMT in HT-29 cell extracts [Pegg et al. (1991) Carcinogenesis 12, 1679-1683] and showed the extracts to be active in conjugation of the MGMT protein with ubiquitin. The proteolysis of O6-BG-inactivated MGMT in HT-29 cell extracts was energy-dependent and was markedly stimulated by ATP and Mg2+ ions. Using the ts85 temperature-sensitive mutant cell line, which expresses a thermolabile ubiquitin-activating enzyme, we observed a differential stability of the inactivated MGMT protein at permissive and nonpermissive temperatures. These results provide conclusive evidence that the MGMT protein, following its inactivation, is degraded via the ubiquitin proteolytic pathway.

Tamoxifen accelerates proteasomal degradation of O6‐methylguanine DNA methyltransferase in human cancer cells

“…Our data show that there was a slight decline in MGMT protein in cells treated with CHX alone, which is consistent with previous finding that wild-type MGMT protein has a long t 1/2 in cells unexposed to alkylating agents. 8,9 In contrast, the t 1/2 of MGMT protein was reduced after tamoxifen treatment (Fig. 5).…”

“…7,8 This action inactivates one MGMT protein molecule for each lesion repaired and makes MGMT a suicide protein, because alkylated MGMT is then degraded through the ubiquitin-dependent proteasomal pathway. 9 The alkylated base adduct is generated in DNA either endogenously or after exposure to alkylating carcinogens and antitumor drugs with methylating and chloroethylating properties, such as chemotherapeutic 2-chloroethyl-N-nitrosourea (CNU) derivatives [e.g., 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)] 8,10 and monofunctional triazenes [e.g., dacarbazine (DTIC)]. 11 Although O 6 -methylguanine is a relatively minor component of methylated base adducts, it is a major mutagenic and carcinogenic lesion because it can pair not only with cytosine but also with thymine during DNA replication, leading to a GC to AT transition mutation.…”

CpG methylation of MGMT and hMLH1 promoter in hepatocellular carcinoma associated with hepatitis viral infection

“…MGMT is converted to an inactive form after removing the methyl group from O 6 -methylguanine (Ishibashi et al, 1994). The inactivated MGMT is not degraded but remains in an immunoreactive state in normal cells (Liu et al, 2001), whereas it is degraded rapidly via the ubiquitin proteolytic pathway in tumour cells (Srivenugopal et al, 1996;Liu et al, 2001). In the light of this, some tumours that correspond to HCCs, 16C -28C, might be regarded as MGMTnegative because of the rapid degradation of the inactive form, although the gene might have been transcribed.…”

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].…”