Role of MGMT in protecting against cyclophosphamide-induced toxicity in cells and animals

Abstract

O(6)-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that protects cells from the biological consequences of alkylating agents by removing alkyl groups from the O(6)-position of guanine. Cyclophosphamide and ifosfamide are oxazaphosphorines used clinically to treat a wide variety of cancers; however, the role of MGMT in recognizing DNA damage induced by these agents is unclear. In vitro evidence suggests that MGMT may protect against the urotoxic oxazaphosphorine metabolite, acrolein. Here, we demonstrate that Chinese hamster ovary cells transfected with MGMT are protected against cytotoxicity following treatment with chloroacetaldehyde (CAA), a neuro- and nephrotoxic metabolite of cyclophosphamide and ifosfamide. The mechanism by which MGMT recognizes damage induced by acrolein and CAA is unknown. CHO cells expressing a mutant form of MGMT (MGMT(R128A)), known to have >1000-fold less repair activity towards alkylated DNA while maintaining full active site transferase activity towards low molecular weight substrates, exhibited equivalent CAA- and acrolein-induced cytotoxicity to that of CHO cells transfected with plasmid control. These results imply that direct reaction of acrolein or CAA with the active site cysteine residue of MGMT, i.e. scavenging, is unlikely a mechanism to explain MGMT protection from CAA and acrolein-induced toxicity. In vivo, no difference was detected between Mgmt-/- and Mgmt+/+ mice in the lethal effects of cyclophosphamide. While MGMT may be important at the cellular level, mice deficient in MGMT are not significantly more susceptible to cyclophosphamide, acrolein or CAA. Thus, our data does not support targeting MGMT to improve oxazaphosphorine therapy.

Figure 1. MGMT expression and activity in transfected CHO lines

MGMT protein expression was determined on 50 μg of whole cell lysates prepared from transfected CHO lines via Western blot analysis. Lane 1, HT29; lane 2, CHO-pcDNA3; lane 3, CHO-MGMT^R128A; lane 4, CHO-wtMGMT. MGMT activity (fmol/mg total protein) was also determined on whole cell lysates. Lysates from HT29 cells were used as positive controls.

Figure 2. Effect of MGMT and MGMT^R128A on cytotoxicity induced by acrolein

CHO lines transfected with pcDNA3, MGMT^R128A, or wtMGMT were treated with acrolein in serum-free media for 4 h. Media was replaced with media containing serum. After 16 h, cytotoxicity was measured as colony-forming ability 9–12 days after treatment. The data represents the mean of three or more separate experiments (except for cytotoxicity determined at 15 μM). Bars are standard deviation. * indicates p < 0.05.

Figure 3. Effect of MGMT and MGMT^R128A on cytotoxicity induced by CAA

CHO lines transfected with pcDNA3, MGMT^R128A, or wtMGMT were treated with 12 or 24 μM CAA in serum-free media. After 4 h drug incubation, the media was replaced with media containing serum. After 16 h, cytotoxicity was measured as colony -forming ability 9–12 days after treatment. The data represents the mean of three or more separate experiments. Bars are standard deviation. ***, ** and * indicate p < 0.003, p < 0.01 and p < 0.03, respectively.

Figure 4. Weight change and deaths in Mgmt +/+ and −/− mice within 14 days following a single intraperitoneal injection with vehicle (0.9% NaCl), cyclophosphamide, acrolein or CAA

Each bar repres ents mean weight change relative to pre-injection for at least 3 mice/group. *Represents death(s) in a treatment group, specifically, 1/6 and 4/6 Mgmt +/+ mice died in the 275 and 300 mg/kg cyclophosphamide group, respectively as well as 2/7 and 1/8 mice died in Mgmt +/+ and Mgmt −/− mice, respectively, treated with 20 mg/kg CAA.

Scheme 1

Essential Elements of the Metabolism of Cyclophosphamide and Ifosfamide