JCTJournal of Cancer Therapy2151-1934Scientific Research Publishing10.4236/jct.2013.44103JCT-32721ArticlesMedicine&Healthcare Expression of O<sup>6</sup>-Methylguanine-DNA Methyltransferase Examined by Alkyl-Transfer Assays, Methylation-Specific PCR and Western Blots in Tumors and Matched Normal Tissue imikoIshiguro1*KrishnamurthyShyam2PhilipG. Penketh2RaymondP. Baumann2AlanC. Sartorelli2ThomasJ. Rutherford2ElenaS. Ratner1Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, New Haven, USADivision of Gynecologic Oncology, Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, USA* E-mail:kimiko.ishiguro@yale.edu(II);310520130404919931April 10th, 2013May 11th, 2013 May 19th, 2013© Copyright 2014 by authors and Scientific Research Publishing Inc. 2014This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/

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.

O<sup>6</sup>-Methylguanine-DNA Methyltransferase (MGMT O<sup>6</sup>-Alkylguanine-DNA Alkyltransferase AGT); [Benzene-<sup>3</sup>H]O<sup>6</sup>-Benzylguanine; Methylation-Specific PCR (MSP); Laromustine (Onrigin Cloretazine VNP40101M 101M); Temozolomide1. Introduction

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 [1]. This conclusion derives from overwhelming evidence that the repair protein O6-methylguanine-DNA methyltransferase (MGMT), which transfers guanine O6- alkyl groups to the active site cysteine in the protein and restores the O-6 position of guanine to the native state, is the principal tumor resistance factor for these agents [1-5].

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 [3]. Although MGMT produces marked tumor resistance to both types of alkylating agents, the underlying mechanisms are different [6]. MGMT repairs the methyl lesions with enormous efficiency until the MGMT pool is exhausted. In contrast, the repair of the chloroethyl lesions by MGMT is complex due to chemical progression of the initial guanine O6-chloroethyl lesion via intermediary N1,O6-ethanoguanine cyclization to the ultimate 1-(N3- cytosinyl)-2-(N1-guaninyl)ethane DNA cross-link. MGMT produces repair of the first two chemical forms [7], but fails to repair the G-C cross-link that does not involve the O-6 position of guanine. Since the cross-link formation competes with the MGMT repair, the repair of the chloroethyl adducts by MGMT is ineffective. The number of guanine O6-methyl and O6-chloroethyl lesions/cell necessary to produce 50% growth inhibition is estimated to be 5,600 and ~300, respectively [6]. Thus, the smaller number of chloroethyl lesions needed for cytotoxicity compensates for the ineffectiveness of MGMT repair, allowing MGMT to produce marked tumor resistance to the chloroethylating agents.

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 [1]. Regeneration of MGMT activity is a slow process that depends upon de novo protein synthesis. Thus, methylating agents such as temozolomide, which require thousands of guanine O6-methylations to cause cytotoxicity, deplete the cellular MGMT pool by providing MGMT with strong substrates. On the other hand, chloroethylating agents, which need only hundreds of guanine O6-lesions for cytotoxicity do not significantly deplete MGMT.

The standard therapy for malignant gliomas is comprised of debulking surgery followed by adjuvant radiotherapy with concomitant temozolomide chemotherapy [8]. Because the brain has the lowest MGMT content among human organs [9,10], brain tumors may generally be susceptible to temozolomide. Moreover, because the daily oral dose of temozolomide at 75 mg/m2 for up to 7 weeks is safe [11], the MGMT-depleting, self-sensitizing property of temozolomide is assumed to enhance its therapeutic activity [8].

Laromustine is a chloroethylating agent designed and synthesized in our laboratory [12]. Although laromustine bears a functional resemblance to the nitrosourea carmustine, laromustine exhibits greater specificity for the O-6 position of guanine than carmustine [6,13]. A clear inverse relationship between the ability of laromustine to produce tumor regression and MGMT activity is observed in CNS tumor xenografts in athymic mice [14]. In phase II clinical studies, laromustine as a single agent produced about a 30% complete response rate with modest extramedullary toxicity in patients with poor-risk acute myelogenous leukemia and myelodysplastic syndromes [15]. The relationship between MGMT activity and clinical response to laromustine remains to be established.

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 [17]. This assay relies upon the covalent transfer of radioactive benzyl moieties from the methanol-soluble small chemical substrate to methanol-insoluble MGMT, and involves incubation of either tissue homogenates or intact cells with the labeled substrate followed by measurement of radioactivity in a 70% methanol-insoluble fraction.

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 [18]. Yet, whether promoter methylation is a tumor specific event has not been tested in strict side-by-side analyses using tumors and matched normal tissue.

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 [20], the organ with the highest MGMT content [9,10]. The occurrence of poor MGMT expression in the absence of promoter methylation has been reported in glioblastoma maintained as xenografts [21] and esophageal squamous cell carcinoma [22]. These observations prompted us to conduct comprehensive analyses on MGMT expression in organs such as the colon, kidney, lung and liver from which pairs of tumors and matched normal adjacent tissue are available through routine resection. We report that 1) promoter methylation is not restricted to tumor, 2) the correlation between promoter methylation and MGMT activity is low, 3) tumor-selective reduction in MGMT activity occurs at a low frequency in the absence of promoter methylation, and 4) tumor-selective reduction of MGMT activity may be mediated by a post-translational mechanism(s).

2. Materials and Methods2.1. Human Tumors and Matched Normal Adjacent Tissue

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.

2.2. Alkyl-Transfer Assays to Measure Functional MGMT Activity

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 [17]. For clinical samples, a fragment weighing 70 - 110 mg was minced with curved iris scissors, suspended in 9 volumes of a buffer consisting of 50 mM Tris-HCl (pH 7.8) and 1 mM dithiothreitol, homogenized on ice using a T-10 Ultra Turrax homogenizer (IKA, Germany), and further sonicated 6 times in short bursts on ice using a Branson Sonifier (Danbury, CT). The final appearance of tissue homogenates was translucent. The homogenate in a volume of 100 µl, in triplicate, was incubated with [3H]-BG in the absence or presence of excess unlabeled O6-benzylguanine and processed as described for intact cultured cells [17]. Final washed pellets were suspended in 400 µl of Biosol (National Diagnostics, Atlanta, GA), transferred to 5 ml-plastic scintillation vials, and incubated at 50˚C for 1 hour in the presence of 20 µl of 30% hydrogen peroxide for solubilization and decolorization. After addition of 4 ml of Bioscint Scintillation Cocktail (National Diagnostics), radioactivity was measured. The protein concentration of tissue homogenates was determined using a Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA) with bovine serum albumin as a standard and MGMT activity was expressed as fmol/mg protein.

2.3. MSP Analysis on Human MGMT Gene Promoter

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.

2.4. Human Tumor Cell Lines

Human tumor cell lines of known MGMT content were described previously [17] except that HeLa cells were purchased from the American Type Culture Collection (Manassas, VA). The MGMT content of HeLa cells was determined using the alkyl-transfer assay [17].

2.5. Western Blot Analyses

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 [25] followed by denaturation at 100˚C for 7 minutes. Cultured cells were washed once with cold phosphate buffered saline and blended in 2× Laemmli’s sample buffer using pipette tips. Tissue fragments were ground in 2× Laemmli’s sample buffer using 8.5 cm-long pestles and 1.5 ml tubes (19923- 0000, Bel-Art Products, South Wayne, NJ).

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).

2.6. Measurement of Signal Intensity

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].

2.7. Statistics

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.

3. Results3.1. The Interrogated Region of the MGMT Gene Promoter for MSP Analyses

Human cell lines have been historically categorized as mer+/− (N-methyl-N'-nitro-N-nitrosoguanidine damage repair) [26] or mex+/− (methyl excision) [27] phenotype according to the resistance/sensitivity to guanine O6- methylating agents until the entity responsible for the phenotype was identified as MGMT. A subset of cultured cell lines completely lacks MGMT expression due to the absence of MGMT mRNA [28]. The promoter region ranging from −480 to +300 relative to the transcription start site (+1) [29] of the human MGMT gene is comprised of a CpG island with 73.8% GC content and 98 CpG sequences (Figure 1(a)), and methylation sensitive restriction endonuclease analyses have established a link between promoter methylation and gene silencing [30]. However, not all CpG sites in the non-expressor promoter are methylated [31]. Bisulfite sequencing applied to the region from −260 to +270 resulted in the identification of “methylation hot spots” (−249 to −103 and +107 to +196; black boxes in Figure 1(a)) in the nonexpressor promoter [32].

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 (Figure 1(b)). This interrogated region coincides with one of the methylation hot spots. These primer sets were employed in this study because of the widespread usage of this interrogated region in glioma studies.

3.2. MGMT Expression Examined by Alkyl-Transfer Assays, MSP and Western Blots in Human Tumor Cell Lines

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 [17]. Using this assay, we previously identified human tumor cell lines with graded MGMT activities ranging from null to 42,000 molecules/cell [17].

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 (Figure 2(a)). In intermediate MGMT expressors such as HL-60 (17,000), NB4 (8,500) and A549 (6,600) cells, both U and M products were generated (Figure 2(a)). The percent of promoter methylation was negatively correlated with MGMT activity (molecules/cell), with the correlation coefficient (r) of −0.93 (Figure 2(c), left).

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 (Figure 2(b), top). The signal intensity of the MGMT protein was positively correlated with MGMT activity, with an r of +0.98 (Figure 2(c), right). Since promoter methylation is linked to epigenetic gene silencing, these results demonstrate the straightforward relationship between gene silencing, MGMT protein expression and functional MGMT activity in human tumor cell lines.

3.3. Functional MGMT Activity in Tumors and Matched Normal Adjacent Tissue

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. Figure 3(a) summarizes MGMT activity expressed as fmol/mg protein in a total of 66 malignant and matched normal tissue samples. Variability in MGMT activity in tumors and normal tissue is shown in the range (R) and mean ± SD (M). The range and mean of MGMT activity in various organs in Figure 3(a) are in agreement with those from the traditional assay using DNA reacted with N-[3H]methylN-nitrosourea [9,10]. It is notable that tumor selective 4- to 5-fold reduction in MGMT activity occurred in K7, K8 and Lv2 (squared and marked with * in Figure 3(a)), albeit at a low frequency (2/12 = 0.17 in the kidney; 3/33 = 0.09 in the combined organs).

Figure 3(b) shows the data rearranged according to the MGMT activity in normal tissue in a column format. MGMT activity in the majority of tumors from the colon

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