Search

The protein O 6-methylguanine-DNA methyltransferase (MGMT) is able to repair the mutagenic O 6-methylguanine (O 6-MeG) adduct back to guanine. In this context, it may protect against colorectal cancer formation associated with N -nitroso compounds. Such compounds may be endogenously formed by nitrosylation of amino acids, which can give rise to mutagenic O 6-MeG and O 6-carboxymethylguanine (O 6-CMG) adducts. It is well established that O 6-MeG is repaired by MGMT. However, up to now, whether O 6 -CMG is repaired by this enzyme remains unresolved. Therefore, the aim of the present study was to analyze the fate of both types of O 6-guanine adducts in the presence and absence of MGMT activity. To this end, MGMT activity was efficiently blocked by its chemical inhibitor O 6-benzylguanine in human colon epithelial cells (HCECs). Exposure of cells to azaserine (AZA) caused significantly higher levels of both O 6-MeG and O 6-CMG adducts in MGMT-inhibited cells, with O 6-CMG as the more abundant DNA lesion. Interestingly, MGMT inhibition did not result in higher levels of AZA-induced DNA strand breaks in spite of elevated DNA adduct levels. In contrast, MGMT inhibition significantly increased DNA strand break formation after exposure to temozolomide (TMZ), a drug that exclusively generates O 6-MeG adducts. In line with this finding, the viability of the cells was moderately reduced by TMZ upon MGMT inhibition, whereas no clear effect was observed in cells treated with AZA. In conclusion, our study clearly shows that O 6-CMG is repaired by MGMT in HCEC, thereby suggesting that MGMT might play an important role as a tumor suppressor in diet-mediated colorectal cancer. [ABSTRACT FROM AUTHOR]

Objectives: Serum is among the agents known to induce hypertrophy of cardiac myocytes, which occurs concomitant with an increase in AP-1-mediated transcription. We have examined if this effect correlates with changes in the relative abundance of particular AP-1 heterodimers, as their exact composition under these conditions is unknown. Furthermore, we obtained insight on the specific role of c-Fos from studying the induction of the glucose transporter GLUT1 by serum in fibroblasts. Methods: We characterised the AP-1 heterodimers expressed in neonatal cardiac myocytes by supershift electrophoretic mobility shift assay (EMSA) analysis. Quantitative changes in transcription were measured using a luciferase reporter vector, and we examined the expression of the glucose transporter GLUT1 in cardiac myocytes and a c-Fos knockout-derived fibroblast cell line by western blotting. Results: Transcriptionally active AP-1 in combinations of c-Jun, JunD and JunB with Fra1, Fra2 and possibly FosB, are expressed in cardiac myocytes. Hypertrophic stimuli transiently induced AP-1 dimers containing c-Fos, and this was dependent on the ERK mitogen-activated protein kinase pathway and coincided with the activation of AP-1-mediated transcription and the induction of GLUT1 in cardiac myocytes. In fibroblasts, the induction of GLUT1 by serum required the specific expression of c-Fos. Conclusion: Our data suggest that induction of c-Fos containing AP-1 heterodimers may partly activate AP-1-mediated transcription in cardiac myocytes treated with hypertrophic agonists under conditions known to induce GLUT1. Data obtained in fibroblasts treated with serum lead us to hypothesise that c-Fos might play a major role in the regulation of GLUT1 expression. [Copyright &y& Elsevier]