Search

Genome wide experiments indicate both proteasome- and vacuole-mediated protein degradation modulate sensitivity to classical DNA-damaging agents. Here, we show that global protein degradation is significantly increased upon methyl methanesulfonate (MMS) exposure. In addition, global protein degradation is similarly increased upon exposure to 4-nitroquinoline- N-oxide (4NQO) and UV and, to a lesser extent, tert-butyl hydroperoxide. The proteasomal inhibitor MG132 decreases both MMS-induced and 4NQO-induced protein degradation, while addition of the vacuolar inhibitor phenylmethanesulfonyl fluoride does not. The addition of both inhibitors grossly inhibits cell growth upon MMS exposure over and above the growth inhibition induced by MMS alone. The MMS-induced protein degradation response remains unchanged in several ubiquitin-proteasome and vacuolar mutants, presumably because these mutants are not totally deficient in either essential pathway. Furthermore, MMS-induced protein degradation is independent of Mec1, Mag1, Rad23, and Rad6, suggesting that the protein degradation response is not transduced through the classical Mec1 DNA damage response pathway or through repair intermediates generated by the base excision, nucleotide excision, or postreplication–DNA repair pathways. These results identify the regulation of protein degradation as an important factor in the recovery of cells from toxicity induced by classical DNA-damaging agents. [ABSTRACT FROM AUTHOR]

O6-methylguanine DNA methyltransferase (MGMT) deficiency is associated with an increased susceptibility to alkylating agent toxicity. To understand the contribution of MGMT in protecting against cyclophosphamide (CP)-induced toxicity, mutagenesis and tumorigenesis, we compared the biological effects of this agent in transgenic Mgmt knockout and wild-type mice. In addition, neurofibromin (Nf1)+/− background was used to increase the likelihood of CP-induced tumorigenesis. Cohorts of Mgmt-proficient or -deficient mice (either Nf1+/+ or Nf1+/−) were given 6 weekly injections of a maximally tolerated dose of CP (250 mg/kg) or vehicle and followed for 15 months. CP-treated mice had more deaths than control mice but there was no difference in the long-term survival between Mgmt+/+ and Mgmt−/− mice (12 of 83 Mgmt+/+ mice died compared to 12 of 80 Mgmt−/− mice, disregarding Nf1 status). Lymphomas and adrenal tumours were the most frequent malignancies. Interestingly, CP-treated, Mgmt-deficient mice developed fewer tumours than controls. Ten of 71 (14%) Mgmt-proficient mice developed tumours after CP treatment compared to only 2 of 68 (3%) Mgmt-deficient mice (P = 0.02). Mgmt−/−, Nf1+/− mice developed fewer tumours (1 of 35, 3%) following CP compared to Mgmt+/+, Nf1+/− mice (7 of 37, 19%) (P = 0.03). Hypoxanthine–guanine phosphoribosyltransferase mutation assays showed no significant increases in mutant frequencies in Mgmt−/− (18.1 × 106) compared to Mgmt+/+ mice (12.9 × 106). These data indicate that MGMT deficiency does not protect against long-term toxicity or mutagenicity from CP and appears to attenuate the occurrence of CP-induced tumours in an Nf1+/− background. [ABSTRACT FROM AUTHOR]

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) protects from toxicity and mutations incurred following alkylating agents by removing O6-alkylguanine lesions. Using Mgmt-/- mice, we examined MGMTs role in protecting from in vivo mutations induced by three different alkylating agents, temozolomide (TMZ), 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) and cyclophosphamide. Mutant frequencies were determined in the hypoxanthine–guanine phosphoribosyltransferase gene of splenic T-lymphocytes from C57BL/6 mice (Mgmt+/+ and Mgmt-/-) following TMZ, BCNU or cyclophosphamide. Following TMZ, the mutation frequency was significantly greater in Mgmt-/- mice (5.5 and 9.8 × 10-6 for 7 and 10 mg/kg TMZ, respectively) compared with vehicle-treated mice (1.0 × 10-6, P = 0.05). In contrast, TMZ-induced mutations were not increased over vehicle in Mgmt+/+ mice. The mutation frequency of mice treated with BCNU (7.5 mg/kg) was the same regardless of Mgmt status. Similarly, pretreatment of Mgmt+/+ mice with 30 mg/kg O6-benzylguanine, a potent inactivator of MGMT, prior to BCNU (15 mg/kg) did not result in significantly more mutations than mice treated with BCNU alone. Following cyclophosphamide, mutation frequencies significantly increased from 1.8 × 10-6 in control-treated mice to 12.9 × 10-6 in Mgmt+/+ and 18.1 × 10-6 in Mgmt-/- mice, although the difference in Mgmt-/- compared with Mgmt+/+ was not significant. Acrolein and chloroacetaldehyde, metabolites of cyclophosphamide, were not mutagenic in Mgmt+/+ and Mgmt-/- mice. These results demonstrate that MGMT significantly protects against in vivo TMZ-induced mutations and that MGMT deficiency does not result in greater mutation frequency following cyclophosphamide or BCNU compared with wild-type mice. [ABSTRACT FROM AUTHOR]