The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity being lower in tumor than in host tissue. Despite the established role of MGMT as a tumor resistance factor, consensus on how to assess MGMT expression in clinical samples is unsettled. The aim of this study is to examine the relationship between the values derived from distinctive MGMT measurements in 13, 12, 6 and 2 pairs of human tumors and matched normal adjacent tissue from the colon, kidney, lung and liver, respectively, and in human cell lines. The MGMT measurements included 1) alkyl-transfer assays using [benzene-3H]O6-benzylguanine as a substrate to assess functional MGMT activity, 2) methylation-specific PCR (MSP) to probe MGMT gene promoter CpG methylations as a measure of gene silencing, and 3) western immunoblots to analyze the MGMT protein. In human cell lines, a strict negative correlation existed between MGMT activity and the extent of promoter methylation. In tissue specimens, by contrast, the correlation between these two variables was low. Moreover, alkyl-transfer assays identified 3 pairs of tumors and normal tissue with tumor-selective reduction in MGMT activity in the absence of promoter methylation. Cell line MGMT migrated as a single band in western analyses, whereas tissue MGMT was heterogeneous around its molecular size and at much higher molecular masses, indicative of multi-layered post-translational modifications. Malignancy is occasionally associated with a mobility shift in MGMT. Contrary to the prevalent expectation that MGMT expression is governed at the level of gene silencing, these data suggest that other mechanisms that can lead to tumorselective reduction in MGMT activity exist in human tissue.
Despite the N-7 position of guanine being the predominant nucleophilic center for DNA alkylation, the antitumor activity of clinically active alkylating agents such as laromustine (onrigin; cloretazine; VNP40101M; 101M), carmustine (BCNU) and temozolomide, is primarily due to their ability to alkylate DNA at the O-6 position of guanine [
MGMT has alternatively been denoted as O6-alkylguanine-DNA alkyltransferase (AGT) in numerous publications. Because human MGMT repairs a variety of guanine O6-alkyl adducts apart from the guanine O6- methyl adduct, AGT reflects the functional property more accurately than MGMT. However, MGMT is used in this paper, because O6-methylguanine-DNA methyltransferase (MGMT) is the name (symbol) approved by the HUGO Gene Nomenclature Committee.
Laromustine and carmustine are chloroethylating agents, while temozolomide is a methylating agent. These two types of alkylating agents exert cytotoxicity through distinctive mechanisms, chloroethylating agents via the generation of highly lethal interstrand DNA cross-links and methylating agents via an intact mismatch repair system [
MGMT acts alone in catalyzing the transfer of an alkyl group to the active site cysteine in a single-step manner that results in a stoichiometric irreversible inactivation of the protein [
The standard therapy for malignant gliomas is comprised of debulking surgery followed by adjuvant radiotherapy with concomitant temozolomide chemotherapy [
Laromustine is a chloroethylating agent designed and synthesized in our laboratory [
Functional MGMT activity determined by an alkyltransfer assay is the logical predictor of response to guanine O6-alkylating agents. However, traditional alkyltransfer assays using DNA reacted with N-[3H]methylN-nitrosourea as a substrate are tedious requiring the use of HPLC in some of the protocols [9,16]. Hence, we have devised a simple alkyl-transfer assay using the pseudosubstrate [benzene-3H]O6-benzylguanine [
MGMT causes tumor resistance to guanine O6-alkylating agents, while it protects normal host tissue from adverse effects. Thus, tumor selectivity by these agents requires differential expression of MGMT in tumor and normal tissue; the lower the MGMT content in tumor and the higher the MGMT content in normal tissue, the greater the tumor selectivity. The obstacles associated with this class of agents are the low occurrence of MGMTnegative or MGMT-low tumors, necessitating rigorous screening for MGMT activity, and a shortage of reliable clinical MGMT assays.
MGMT gene promoter methylation examined by methylation-specific PCR (MSP) has emerged as an independent prognostic marker, as well as a predictive marker for response to temozolomide in malignant gliomas [18,19]. MSP yields an indirect measure of MGMT expression. Thus, to be a predictive marker for drug response, promoter methylation must be validated for correlation with endpoint MGMT activity. The MSP assay is stated to rely upon the fact that detection of the methylated MGMT allele can be solely attributed to neoplastic cells and nontumor tissue contamination of the surgical specimen does not interfere with the result [
Using pairs of tumors and matched normal tissue, the occurrence of tumor specific absence or reduction in MGMT expression has been reported in the liver [
Snap frozen tissue samples were obtained from the Eastern Division of the Cooperative Human Tissue Network (CHTN), a National Cancer Institute supported resource. The application requesting samples from the CHTN was reviewed by the Yale University Human Investigation Committee and received non-human investigation status. Each sample was accompanied by unidentifiable information (age, sex, race and pathology report) and a hematoxylin-eosin stained tissue slide. Tumors were accepted only when matched normal adjacent tissue was available with a minimum weight of 0.1 g to enable preparation of homogenates for alkyl-transfer assays. From August 2010 through August 2011, we received 13, 12, 6, and 2 sets of tumor and normal tissue samples from the colon, kidney, lung and liver, respectively, with 8 separate deliveries.
The assay procedures for intact cultured cells and cell homogenates using [benzene-3H]O6-benzylguanine ([3H]- BG, 23.6 Ci/mmol, MT1915, Moravek Biochemicals, Brea, CA) were previously described [
Genomic DNA was extracted either from 5 - 15 mg of solid tissue or from 2 × 106 cultured cells using a Gentra Puregene DNA purification kit (QIAGEN, Germany) according to the manufacturer’s manual. Purified DNA was quantified using a TBS-380 mini-fluorometer (Turner BioSystems, Sunnyvale, CA) using Hoechst 33258 dye and calf thymus DNA as a standard according to the manufacturer’s protocol. DNA (2.5 µg) was subjected to a bisulfite conversion reaction using an EpiMark bisulfite Conversion kit (New England Biolabs, Inc., Ipswich, MA) according to the manufacturer’s instruction manual. For PCR, sets of primer sequences described by Esteller et al. [23,24] were employed: for the unmethylated reaction; 5’-TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3’(U- 93-F) and 5’-AACTCCACACTCTTCCAAAAACAAA ACA-3’(U-93-R) and for the methylated reaction; 5’- TTTCGACGTTCGTAGGTTTTCGC-3’ (M-81-F) and 5’- GCACTCTTCCGAAAACGAAACG-3’ (M-81-R). The PCR reaction mixture consisted of 4 µl of the bisulfite modified DNA eluate (40 µl), 1× PCR reaction buffer, 0.2 mM dNTP mixture, 0.2 µM each forward and reverse primer, and 1 unit of TaKaRa Taq HS (Takara Bio Inc., Japan) in a volume of 25 µl. The thermocycling protocol consisted of 40 cycles of 95˚C for 45 seconds, 60˚C for 45 seconds and 72˚C for 60 seconds. PCR products were subjected to 3% MetaPhor agarose (Cambrex Bio Science, Rockland, ME) horizontal gel electrophoresis with TBE buffer containing 0.5 µg/ml of ethidium bromide.
Human tumor cell lines of known MGMT content were described previously [
Two procedures were employed for sample preparation. The first procedure involved solubilization of intact cultured cells (5 × 106 cells) or tissue fragments (20 mg) in 0.25 ml of 2× Laemmli’s sample buffer [
In the second procedure, cultured cells were washed once with cold phosphate buffered saline, suspended at a density of 5 × 107 cells/ml in 50 mM Tris-HCl (pH 7.5) buffer in the presence or absence of 1× Halt Protease & Phosphatase Inhibitor Cocktail (78440, Thermo Scientific, Rockford, IL), and sonicated 4 times in short bursts on ice. Tissue homogenates were prepared as described in the alkyl-transfer assay in 50 mM Tris-HCl (pH 7.5) buffer in the absence or presence of the cocktail of protease and phosphatase inhibitors. The homogenates from cultured cells or tissue fragments were mixed with an equal volume of 2× Laemmli’s sample buffer and denatured at 100˚C for 7 minutes.
The tissue or cell homogenates (80 µg of protein/lane), whole tissue extracts (20 µl/1.6 mg tissue/lane), and whole cell extracts (20 µl/5 × 104 cells/lane), were resolved by 0.1% SDS-10% or 12.5% PAGE. Following conventional western procedures, chemiluminescent images were captured using G:Box iChemi XR (Syngene, Frederick, MD). Mouse monoclonal anti-human MGMT antibody (clone MT 3.1) was from Millipore (Temecula, CA). Rabbit polyclonal anti-human MGMT antibody (ab69629) was from Abcam (Cambridge, MA). Goat polyclonal anti-human MGMT antibody (AF3794) was from R&D Systems, Inc. Rabbit polyclonal anti-ubiquitin antibody (sc-9133) and goat polyclonal anti-HSC 70 antibody (sc-1059) were from Santa Cruz Biotechnology (Santa Cruz, CA).
Signal intensities of images from MSP and western blots were measured using ImageJ (rsbweb.nih.gov/ij/) according to the formula: [(mean brightness of the selected area—mean brightness of the background of an equal area) × the area].
The strength of the linear relationship between two variables was quantified using Pearson’s coefficient of correlation (r,) where the values 1 and −1 represent perfect positive and negative correlations, respectively, and the value 0 represents no linear correlation.
Human cell lines have been historically categorized as mer+/− (N-methyl-N'-nitro-N-nitrosoguanidine damage repair) [
The unmethylated and methylated primer sets described by Esteller et al. [23,24] amplify from +110 to +202 and from +116 to +196, giving rise to 93 and 81 bp of unmethylated and methylated PCR products, respectively (
Alkyl-transfer assays using [benzene-3H]O6-benzylguanine (3H-BG) enable incubation of intact cultured cells with the labeled substrate, and readily assess MGMT activity as the number of functional MGMT molecules/ cell [
To examine whether epigenetic gene silencing by promoter methylation accounted for the variability, MSP consisting of sodium bisulfite conversion of genomic DNA followed by PCR amplification of the interrogated region using primer sets specific for unmethylated and methylated DNA, was conducted. The unmethylated 93-bp PCR product was predominantly generated in high expressors (42,000 MGMT molecules/cell) such as DU145 and HeLa cells, whereas the methylated 81-bp product was predominantly generated in MGMT null cell lines such as TF-1, U-937 and U251 cells (
MSP analyses do not clarify whether the MGMT locus is hemi-methylated or the MGMT locus is partially methylated on both alleles in intermediate MGMT expressors. NB4 and A549 cells with substantial methylation signal output are highly resistant (17- and 11-fold, respectively) to temozolomide measured under MGMTintact and MGMT-abrogated conditions (data not shown), indicating that the presence of methylation signals per se does not guarantee sensitivity to temozolomide.
The MGMT protein in cell lines migrated as a single band in western analyses (
For simpler presentation, sets of malignant (m) and matched normal (n) adjacent tissue from the colon (C), kidney (K), lung (Lg) and liver (Lv) were renamed and chronologically renumbered. Alkyl-transfer assays used to measure MGMT activity in clinical samples relied upon covalent transfer of radioactive benzyl moieties from 3H-BG to MGMT. After incubation of tissue homogenates with 3H-BG, 70% methanol precipitates containing 3H-benzylated MGMT were thoroughly washed to remove unreacted 3H-BG.