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. 2014 Mar;53(3):201-10.
doi: 10.1002/mc.21964. Epub 2012 Oct 12.

Tumor-associated mutations in O⁶ -methylguanine DNA-methyltransferase (MGMT) reduce DNA repair functionality

Kristy L Lamb  1 , Yanfeng Liu, Kimiko Ishiguro, Youngho Kwon, Nicolas Paquet, Alan C Sartorelli, Patrick Sung, Sara Rockwell, Joann B Sweasy
Affiliations

Tumor-associated mutations in O⁶ -methylguanine DNA-methyltransferase (MGMT) reduce DNA repair functionality

Kristy L Lamb et al. Mol Carcinog. 2014 Mar.

Abstract

MGMT is the primary vehicle for cellular removal of alkyl lesions from the O-6 position of guanine and the O-4 position of thymine. While key to the maintenance of genomic integrity, MGMT also removes damage induced by alkylating chemotherapies, inhibiting the efficacy of cancer treatment. Germline variants of human MGMT are well-characterized, but somatic variants found in tumors were, prior to this work, uncharacterized. We found that MGMT G132R, from a human esophageal tumor, and MGMT G156C, from a human colorectal cancer cell line, are unable to rescue methyltransferase-deficient Escherichia coli as well as wild type (WT) human MGMT after treatment with a methylating agent. Using pre-steady state kinetics, we biochemically characterized these variants as having a reduced rate constant. G132R binds DNA containing an O⁶ -methylguanine lesion half as tightly as WT MGMT, while G156C has a 40-fold decrease in binding affinity for the same damaged DNA versus WT. Mammalian cells expressing either G132R or G156C are more sensitive to methylating agents than mammalian cells expressing WT MGMT. G132R is slightly resistant to O⁶ -benzylguanine, an inhibitor of MGMT in clinical trials, while G156C is almost completely resistant to this inhibitor. The impared functionality of expressed variants G132R and G156C suggests that the presence of somatic variants of MGMT in a tumor could impact chemotherapeutic outcomes.

Keywords: DNA repair; MNNG; O(6)-alkylguanine DNA alkyltransferase; O(6)-benzyguanine; O-(6)-methylguanine.

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Figures

Figure 1
Figure 1
MGMT G132R and G156C are near the active site of MGMT. MGMT bound to the minor groove of DNA (black, bottom center), with one DNA base flipped into the MGMT active site (acceptor residue at CYS145) [24]. Dotted lines represent measured distances, numbers are distances measured in Ångströms (Å). Mutations to cysteine and arginine could result in residues larger than glycine and may distort the structure. Generated in MacPyMol using the 1T38 crystal structure [24].
Figure 2
Figure 2
MGMT G132R and G156C do not fully rescue methyl-transferase deficient E. coli. (A) Western blot for MGMT expression in GWR111 E. coli shows the ratio of WT:G132R:G156C expression to be 1:11.0:11.2. There is no discernable MGMT expression in empty vector cells. (B) Survival curves for E. coli. Log-phase GWR111 cultures expressing wild type (■) or variant (● G132R, ▽G156C) human MGMT and the empty pBAD expression vector (✖) were incubated with graded doses of MNNG for 30 min then plated in serial dilutions. Surviving colonies were counted the next day. Representative survival curves are shown.
Figure 3
Figure 3
G132R and G156C do not change the overall structure of MGMT. Multiple circular dichroism spectra for WT, G132R, and G156C purified human MGMT were measured, averaged, and normalized. The overall shapes of the spectra, with peaks of intensity at 208 and 217 nm, were the same for all three proteins.
Figure 4
Figure 4
MGMT variants have decreased affinity for substrate. (A) Schematic of substrates and the cutting ability PvuII on those substrates. (B) Gel showing separation of repaired and unrepaired products of time course for 33 nM of G132R with 5.3 nM substrate. Time is given in seconds. U is undamaged control substrate. (C) Determination of pre-steady state kobs for G132R. Fraction of substrate repaired is plotted against time. The scale of the X-axis changes at 100 s to allow visualization of points in the early timepoints of the curve, before saturation. 5.3 nM substrate was reacted with varying concentrations (○ 500 nM, ▲ 333 nM, * 33 nM, ◆25 nM, + 5 nM, ■ 2 nM) of G132R MGMT in separate time courses. Repair was evaluated via gel electrophoresis followed by quantification on a phosphorimager and normalized to a no damage control. A single exponential equation was fit to the plot to yield a kobs for each concentration. (D) Single hyperbolic plot to determine Kd and kr for MGMT G132R. The kobs values as calculated in (A) were plotted against nM concentration of MGMT and fit to a single hyperbolic equation to determine the reaction rate (kr) and the binding efficiency (Kd).
Figure 5
Figure 5
MGMT G132R and G156C cannot fully rescue MGMT-deficient mammalian cells. (A) Expression of the variants in EMT6 cells. The ratio of WT:G132R:G156C expressed in these pools is 1:1.3:0.7. (B) Survival of EMT6 cells after treatment with MNNG. EMT6 mouse mammary carcinoma cells transfected with empty pRVY expression vector (×), pRVY containing WT MGMT (■), G132R MGMT (●), or G156C MGMT (▽) were treated with graded doses of MNNG and plated over a range of dilutions. Colonies were stained 10 d later and counted. Points are geometric means from three separate experiments on pools within a range of three passages and error bars represent the standard error of the mean. * P = 0.03 and P = 0.04 for WT versus G132R and G156C, respectively, paired t-test.
Figure 6
Figure 6
G156C does not react with O6BG. 2 × 106 EMT6 mouse mammary carcinoma cells were incubated with tritated benzylguanine. Free benzylguanine was removed by washing with methanol and the amount of tritium transferred to cells was measured by scintillation counting. Averages and standard deviations for triplicate assays are shown. **P < 0.001; P < 0.0001.
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