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Mol Cancer Ther. 2006;5:121-128
© 2006 American Association for Cancer Research

Differential competitive resistance to methylating versus chloroethylating agents among five O6-alkylguanine DNA alkyltransferases in human hematopoietic cells

Aparecida Maria Fontes1, Brian M. Davis1, Lance P. Encell2, Karen Lingas1, Dimas Tadeu Covas3, Marco Antonio Zago3, Lawrence A. Loeb4, Anthony E. Pegg5 and Stanton L. Gerson1

1 Division of Hematology/Oncology and Comprehensive Cancer Center, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio; 2 Promega Corporation, Madison, Wisconsin; 3 Regional Blood Center and Faculdade de Medicina de Ribeirão Preto da Universidad de São Paulo, Centro de Terapia Celular, Ribeirão Preto, Brazil; 4 The Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington School of Medicine, Seattle, Washington; and 5 Department of Cellular and Molecular Physiology, Penn State Medical Center, Hershey, Pennsylvania

Requests for reprints: Stanton L. Gerson, Division of Hematology Oncology, BEB-3 Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4955. Phone: 216-368-1177; Fax: 216-368-1166. E-mail: slg5{at}po.cwru.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
P140K-MGMT and G156A-MGMT genes encode two O6-benzylguanine–resistant O6-alkylguanine DNA alkyltransferase proteins that confer a high degree of O6-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or O6-benzylguanine and temozolomide resistance to primary hematopoietic cells. In this study, we directly compared these and three other O6-benzylguanine–resistant MGMT genes for their ability to protect the human erythroleukemia cell line, K562, using a direct competitive selection strategy to identify the mutation that conferred the greatest degree of protection from O6-benzylguanine and either BCNU or temozolomide. MFG retroviral vector plasmids for each of these mutants [G156A-MGMT (ED50 for O6-benzylguanine, 60 µmol/L); and P140K-MGMT, MGMT-2 (S152H, A154G, Y158H, G160S, L162V), MGMT-3 (C150Y, A154G, Y158F, L162P, K165R), and MGMT-5 (N157T, Y158H, A170S; ED50 for benzylguanine, >1,000 µmol/L)] were mixed, and the virus produced from Phoenix cells was transduced into K562 cells. Stringent selection used high doses of O6-benzylguanine (800 µmol/L) and temozolomide (1,000 µmol/L) or BCNU (20 µmol/L) administered twice, and following regrowth, surviving clones were isolated, and the MGMT transgene was sequenced. None of the mutants was lost during selection. Using temozolomide, the enrichment factor was greatest for P140K-MGMT (1.7-fold). Using BCNU selection, the greatest enrichment was observed with MGMT-2 (1.5-fold). G156A-MGMT, which is the least O6-benzylguanine–resistant MGMT gene of the mutants tested, was not lost during selection but was selected against. The optimal mutant MGMT useful as a drug resistance gene may depend on whether a methylating or chloroethylating agent is used for drug selection. [Mol Cancer Ther 2006;5(1):121–8]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
O6-benzylguanine, an inhibitor of the DNA repair protein, O6-alkylguanine DNA alkyltransferase (AGT) plus 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or O6-benzylguanine plus temozolomide have increased therapeutic efficacy over the chemotherapeutic agents alone in animal tumor xenograft models (1–8). Thus, these combinations are currently being evaluated in clinical trials with cutaneous lymphomas, malignant melanoma, brain neoplasms, multiple myeloma, and gastrointestinal carcinomas (9–12). However, the damage caused to the bone marrow by these alkylating agents manifests in the short term as myelosuppression or pancytopenia, the dose-limiting toxicity in clinical trials, and has the potential to induce therapy-related myelodysplasia or acute leukemia (13, 14).

Mutations in human MGMT have been developed, which retain AGT activity but are O6-benzylguanine resistant. Based on the crystal structure of the AGT active site pocket, it is apparent that these mutants affect the size, hydrophobicity, and flexion of the active site pocket, resulting in exclusion of the benzyl moiety from the active site or a reduction in its ability to undergo an alkyl transfer reaction with the active site cysteine. G156A-MGMT, located within the DNA-binding wing flanking the alkyl-binding pocket (15), is moderately O6-benzylguanine resistant (EC50 = 60 µmol/L). It distorts the adjacent loop (residues 158–160) that constitutes one wall of the benzyl-binding pocket (16, 17). The P140K-MGMT mutant alters the floor of the alkyl-binding pocket, excluding the benzyl group and resulting in a very high degree of O6-benzylguanine resistance (EC50 > 1.2 mmol/L; refs. 18, 19). Molecular evolution with selection allowed the identification of multiple MGMT mutants highly resistant to O6-benzylguanine, which retain the AGT ability to remove alkyl adducts from the O6-guanine position in DNA (20). Three mutants (i.e., MGMT-2, MGMT-3, and MGMT-5), each containing three to five amino acid substitutions in the region of the active site, were the most resistant to O6-benzylguanine and BCNU (EC50 > 2 mmol/L; ref. 20).

These mutants, O6-benzylguanine–resistant MGMT genes, provide a potent selection strategy for drug resistance gene transfer. We have used this for hematopoietic stem cell selection because two chemotherapeutic agents, BCNU, and methylating agents, such as temozolomide, are stem cell toxins, resulting in cumulative stem cell loss and delayed myelosuppression. We first introduced the use of O6-benzylguanine–resistant G156A-MGMT to selectively protect the human CD34 cells and suggested that this selective protection could occur while sensitizing the tumor to chemotherapy using O6-benzylguanine (21). Selecting the best O6-benzylguanine–resistant MGMT for gene therapy is an important therapeutic decision. These mutants vary in stability, level of O6-benzylguanine resistance, and repair capacity for either O6-methylguanine or O6-chloroethylguanine. The aim of the present study was to compare the ability of five of the most potent MGMT mutants to protect human hematopoietic cells against toxicity from O6-benzylguanine + BCNU or O6-benzylguanine + temozolomide treatment using a direct competitive selection strategy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
BCNU and temozolomide were obtained from the Developmental Therapeutics Branch, National Cancer Institute (Bethesda, MD). The BCNU was solubilized in 100% ethanol before dilution in PBS and used within 10 minutes of reconstitution. Temozolomide was solubilized in DMSO (Sigma, St. Louis, MO) before dilution in PBS and was used within 10 minutes of reconstitution. O6-benzylguanine was synthesized by R. Moschel at the Frederick Cancer Research Institute and was solubilized in DMSO. Complete medium for K562 (human chronic myelogenous leukemia cell line) consisted of Iscove's modified Dulbecco's medium (Life Technologies/Bethesda Research Laboratories, Gaithersburg, MD) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), 1% L-glutamine plus 1% penicillin/streptomycin (Life Technologies/Bethesda Research Laboratories); and for producer cell line Phoenix, consisted of DMEM (Life Technologies/Bethesda Research Laboratories) supplemented with 10% fetal bovine serum (Hyclone Laboratories), 1% L-glutamine plus 1% penicillin/streptomycin. Human interleukin-3 and human granulocyte macrophage colony-stimulating factor were purchased from R&D Systems (Minneapolis, MN). The monoclonal antibody anti-MGMT (monoclonal antibody clone MT3.1) was purchased from Kamiya Biomedical Co. (Seattle, WA), and R-phycoerythrin–conjugated antibodies were purchased from Caltag Laboratories (Burlingame, CA). The mouse isotype IgG1 was purchased from Becton Dickinson (San Jose, CA).

Retroviral Vectors
Retroviral vector pMFG-G156A was constructed to express cDNA sequence for G156A-MGMT as previously described (15). Retroviral vector pMFG-P140K was constructed to express cDNA sequence for P140K-MGMT by Davis et al. (22) and was originally selected for dual resistance to N-methyl-N'-nitro-N-nitrosoguanidine and O6-benzylguanine from a library of AGT mutants containing a random sequence at positions 138 to 140 (18). Retroviral vectors pMFG-MGMT-2, pMFG-MGMT-3, and pMFG-MGMT-5, containing the following mutations of the MGMT gene, as previously described, MGMT-2 (S152H, A154G, Y158H, G160S, L162V), MGMT-3 (C150Y, A154G, Y158F, L162P, K165R), and MGMT-5 (N157T, Y158H, A170S), were subcloned into MFG retroviral vector (20). These three MGMT mutant genes were originally selected for dual resistance to N-methyl-N'-nitro-N-nitrosoguanidine and O6-benzylguanine from a library of MGMT mutant genes containing a random sequence at positions 150 to 172 (19) and conferred the greatest degree of resistance to O6-benzylguanine and BCNU in K562 cells (20).

Cell Transfections and Transductions
DNA from each retroviral vector plasmid was isolated using NucleoBond Plasmid Max kit from Clontech (Palo Alto, CA) and then pooled by mixing 2 µg of each construct. A total of 10 µg of retroviral plasmid was transfected into 5 x 106 Phoenix amphotropic packaging cells (a gift from G. Nolon, Stanford University, Stanford, CA), using a calcium phosphate coprecipitation protocol.4 To transduce K562 cells, these amphotropic producer cells were grown to confluence, and the virus was harvested in DMEM supplemented with 10% heat-inactivated fetal bovine serum. To remove contaminating producer cells, supernatant was filtered through a 0.45-µm filter (Millipore, Bedford, MA). Polybrene was added at 5.5 µg/mL. K562 cells were infected twice at intervals of 24 hours. The efficiency of transfection and transduction was measured by flow cytometry.

Flow Cytometric Analysis
To determine the MGMT expression in transduced cells, 1 x 106 cells were washed in 2% bovine serum albumin (BSA)/PBS, stabilized for 30 minutes at 4°C using 1% paraformaldehyde (in PBX 1x), and permeabilized by incubating in 1% Tween 20 (in 2% BSA/PBS) for 30 minutes at 37°C. After permeabilization, cells were washed in 2% BSA/PBS, and nonspecific binding sites were blocked for 30 minutes at 22°C with 10% normal goat serum. AGT antibody mT3.1 (1 µg) was added, and cells were incubated at 4°C overnight. Cells were washed twice with 2% BSA/PBS and incubated with secondary antibody (goat anti-mouse IgG-1{gamma} phycoerytrin conjugated) for 1 hour at 4°C. After washing, cells were resuspended in 300 µL of 2% BSA/PBS for fluorescence-activated cell sorting analysis. Flow cytometry analysis of 10,000 events was done on a FACScan (Becton Dickinson) running CellQuest data acquisition and analysis software (Becton Dickinson). Light scatter was used for gating on permeabilized cells.

Stringent Selection Protocol
High-stringency O6-benzylguanine and BCNU and O6-benzylguanine and temozolomide conditions were used to select O6-benzylguanine–resistant MGMT mutant expressing K562 cells. For O6-benzylguanine and BCNU selection, transfected K562 cells (30 x 106) were treated with 800 µmol/L O6-benzylguanine for 1 hour followed by treatment with 20 µmol/L BCNU for 2 hours. For O6-benzylguanine and temozolomide selection, transfected K562 cells (30 x 106) were treated with 800 µmol/L O6-benzylguanine for 1 hour followed to treatment with 1,000 µmol/L temozolomide for 2 hours. Temozolomide-treated cells were washed in PBS and exposed additionally to 800 µmol/L O6-benzylguanine for 16 to 24 hours to provide the maximal toxicity to residual O6-methylguanine lesions. Cells treated with both agents were washed with PBS to remove the drugs and left to grow for 2 weeks in the presence of 25 µmol/L O6-benzylguanine. This provides ongoing AGT inhibition and is the maximal amount of O6-benzylguanine that can be added without slowing cell cycle times (3). After recovery, a second identical treatment was done. Outgrowth before analysis was for 14 days.

Analysis of the Cytotoxic Effects of O6-Benzylguanine and Temozolomide
Colony-Forming Unit Assay. The O6-benzylguanine and BCNU and O6-benzylguanine and temozolomide resistance conferred to K562 cells by these five O6-benzylguanine–resistant MGMT mutants was determined by clonogenic assays. Transfected K562 cells were exposed to increasing doses of temozolomide or BCNU, in the presence or absence of 25 µmol/L O6-benzylguanine. After treatment, cells were washed in PBS, and 1,000 cells/mL were plated in triplicate in complete methylcellulose medium (0.8% methylcellulose, 1% BSA, 2 mmol/L L-glutamine, 0.1 mmol/L 1-mercaptoethanol, 10 ng/mL human interleukin-3, and 85 ng/mL human granulocyte macrophage colony-stimulating factor in Iscove's modified Dulbecco's medium; Stem Cell technologies, Vancouver, British Columbia, Canada). One-milliliter samples of the mixture were plated into 35-mm dishes and grown for 10 days. Colonies containing >50 cells was enumerated and compared with parental K562 cells.

PCR Analyses. In four independent experiments, individual colonies picked from methylcellulose cultures were washed with PBS, centrifuged, and then resuspended in 25 to 30 µL of a buffer containing 50 mmol/L Tris-HCl (pH 8), 10 mmol/L EDTA, 100 mmol/L NaCl, and 1% Triton X-100, plus 1 mg/mL proteinase K. The digestion was carried out at 55°C for 2 hours. Proteinase K was inactivated by heating at 95°C for 8 to 10 minutes. Two to five microliters of the digested sample were used for PCR analysis. Amplification was done (after an initial 3 minutes at 95°C) for 35 cycles of 30 seconds at 94°C, 40 seconds at the indicated annealing temperature, and 40 seconds at 72°C. A final extension period of 10 minutes was done. Annealing temperature was 62°C for the MGMT gene and 60°C for the dystrophin gene. Primers for MGMT gene (5'-CTTCACCATCCCGTTTTCCAG-3' and 5'-CTGCCAGACCTGAGCTCCCTC-3') amplify a 316-bp product; dystrophin primers (5'-TCACTTGCTTGTGCGCAGG-3' and 5'-GAAAAGTGTATATCAAGGCAGCGACGATAA-3') amplify a 500-bp product. The PCR was done with 40 pmol of each primer, 0.2 mmol/L deoxynucleotide triphosphates, 2 units of Taq polymerase, in a total volume of 50 µL. The PCR products were separated on 1.3% agarose gel and visualized after ethidium bromide staining. The amplified DNA was purified using the QIAquick PCR Purification kit (Qiagen, Valencia, CA) and sequenced to characterize the MGMT gene mutant. Sequence data were analyzed by the MacVector program, version 3.1. The wild-type human MGMT is from Genbank (accession no. NM_002412).

Characterization of the Transduced K562 Cell Population
Analysis of Individual K562 Clones. Individual colonies of transduced K562 cells were picked from methylcellulose and cultured in Iscove's modified Dulbecco's medium as above, and cells were harvested for PCR analysis to evaluate the efficiency of the integration of retrovirus DNA. PCR-positive clones were submitted to fluorescence-activated cell sorting analysis for AGT protein expression as above and to reverse transcription-PCR for mRNA MGMT expression level.

Reverse Transcription-PCR Assay. Total cellular RNA was extracted from 1 x 106 K562 cells with SV total RNA isolation system (Promega, Madison, WI) according to the manufacturer's protocol. Reverse transcription of RNA to cDNA was done according to the protocol of the SuperScript First Strand Synthesis System for reverse transcription-PCR (Life Technologies Bethesda Research Laboratories). For detection of MGMT mRNA, each K562 clone, 10% of the reverse transcriptase reaction with or without reverse transcriptase enzyme, was amplified by PCR with the same thermal cycling profile as above, using 25 cycles. Each PCR was carried out in 25 µL and resolved on a 1.3% agarose gel.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Engineering Erythroleukemia K562 Cells to Constitutively Produce Five Mutant O6-Benzylguanine–Resistant Human Alkyltransferase Proteins
The five O6-benzylguanine–resistant MGMT mutants used in this study (Table 1 ) have different amino acid substitutions. The three mutants containing three to five amino acid changes near the active site and P140K-MGMT have millimolar EC50 values for O6-benzylguanine inactivation, compared with the wild-type value of 0.1 µmol/L O6-benzylguanine (Table 2 ). There is variability among the mutants in protein stability: G156A-MGMT is the least stable.


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  		Table 1. Mutations in O6-benzylguanine resistant MGMTs

 

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  		Table 2. Features of O6-benzylguanine–resistant MGMT proteins

 
For transduction into K562 cells, we generated an amphotropic producer cell line by transfecting Phoenix cells with a pool of the five plasmids (pMFG-G156A-MGMT, pMFG-P140K-MGMT, pMFG-MGMT-2, pMFG-MGMT-3, and pMFG-MGMT-5) mixed at equimolar ratios. Twenty-four hours after transfection, 24% of Phoenix+ cells expressed these transgenes as determined by flow cytometry using an anti-MGMT antibody (data not shown). After two cycles of transduction, the percentage of K562 cells expressing AGT by flow cytometry was 11% in two independent experiments (data not shown).

MGMT Expression in Clones of Transduced K562 Cells
To understand the results of the competitive selection, we determined the proportion of clones expressing MGMT. K562 clones positive for MGMT by PCR were expanded and MGMT expression analyzed by flow cytometric and reverse transcription-PCR assay. Of 14 MGMT+ colonies analyzed by this method, three expressed high levels of AGT protein (89–96.2%, Fig. 1C–E ), one moderate level (30%, Fig. 1F), four low level (4–7%, Fig. 1G–J), and six clones expressed at very low levels or not at all (1–2%, Fig. 1K–P). This analysis was repeated thrice with a total of 48 isolates, and similar results were obtained. Overall, 22% of PCR-positive clones expressed high levels of AGT protein, about 28% expressed low levels, and 43% had undetectable levels of expression.


Figure 1
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  		Figure 1. Alkyltransferase protein expression in MGMT+ K562 clones. A, representative fluorescence-activated cell sorting dot blot light forward scatter/side scatter profile of K562 cells. B, representative histogram of K562 cells incubated with isotype-matched control antibody to establish profile setting. C to P, histograms showing AGT expression for each MGMT+ K562 clone incubated with monoclonal antibody anti-MGMT (monoclonal antibody clone MT3.1). Q and R, histograms showing AGT expression from parental K562 (negative control) and MGMT+/K562 cells known to express high level of this gene, respectively. Percentage of expression for each clone. Top right, % cells expressing AGT.

 
We next asked whether differences in AGT expression were due to transcriptional activity effects or post-transcriptional events. Reverse transcription-PCR analysis of mRNA showed that the highest level of MGMT mRNA was detected in the clones that express high levels of AGT protein (Fig. 2, lanes C1, C37, and C39 ). Much lower levels of MGMT mRNA (Fig. 2, lanes C5, C18, C20, and C32) were detected for the clones that express 7% to 30% of AGT protein (Fig. 1, C5, C18, C20, and C32), whereas the absence of MGMT mRNA (Fig. 2, lanes C4, C6, C22, C24, C26, C27, and C31) was observed in clones with low or absent AGT protein (Fig. 1, C4, C6, C22, C24, C26, C27, and C31). Therefore, the absence of AGT protein is due to silencing of transcriptional activity and not due to translational effects.


Figure 2
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  		Figure 2. Expression MGMT mRNA in K562 MGMT+ clones. Total RNA was prepared from each K562 clone and analyzed by reverse transcription-PCR for MGMT mRNA. Reactions were done without reverse transcriptase (–) to control for DNA contamination.

 
Competitive Selection of Drug-Resistant K562 Clones by Stringent Drug Treatment
MGMT-transduced K562 cells were treated with 800 µmol/L O6-benzylguanine for 1 hour and either 1,000 µmol/L temozolomide or 20 µmol/L BCNU for 2 hours. Surviving cells were retreated with the same drugs. Two independent experiments were done, and the selection results were pooled. Survival after the first drug selection was about 3% of control, with greater survival after the second treatment. To determine the degree of drug resistance conferred to K562 cells by these mutants after high-stringency selection, we determined the BCNU and temozolomide IC90 by seeding cells into methylcellulose medium and monitored for colony formation. K562 cell cultures containing unselected AGT mutants were 2-fold more resistant to BCNU (IC90 of 22 µmol/L) compared with parental K562 cells (IC90 around 10 µmol/L). After high-stringency O6-benzylguanine and BCNU selection, K562 cells were 8-fold more resistance to BCNU (BCNU IC90 nearly 80 µmol/L; Fig. 3 ). Cells treated with BCNU alone had a similar survival curve, presumably because all O6-benzylguanine–resistant MGMTs conferred O6-benzylguanine resistance to the cell cultures, and a difference between the mutants could not be identified in the survival curves. Cells exposed to temozolomide had increased drug resistance after selection (Fig. 4 ). Parental K562 cells had a temozolomide IC90 < 100 µmol/L, whereas transduced K562 cells had a temozolomide IC90 of 600 µmol/L. After high-stringency O6-benzylguanine and temozolomide selection, the temozolomide IC90 increased to >2,000 µmol/L, 20-fold above untransduced cells. When pretreated with 25 µmol/L benzylguanine, there is no significant difference in the temozolomide IC90 value.


Figure 3
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  		Figure 3. Comparison of the O6-benzylguanine (O6-BG) and BCNU resistance conferred to MGMT-transduced K562 cells before and after high-stringency selection. K562 cells were treated with BCNU alone ({square}, without selection; {blacksquare}, after selection) or O6-benzylguanine and BCNU ({triangleup}, without selection; {blacktriangleup}, after selection). Cells were exposed to 25 µmol/L O6-benzylguanine for 1 h followed with 0 to 80 µmol/L of BCNU. As a control, we measured the sensitivity of parental K562 cells to BCNU (bullet). K562 cells were plated at 1,000 per dish. After 10 to 12 d, colonies were enumerated.

 

Figure 4
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  		Figure 4. Comparison of the O6-benzylguanine (O6-BG) and temozolomide (TMZ) resistance conferred to MGMT-transduced K562 cells before and after high-stringency selection. K562 cells were treated with temozolomide alone ({square}, without selection; {blacksquare}, after selection) or O6-benzylguanine and temozolomide ({triangleup}, without selection; {blacktriangleup}, after selection). Cells were exposed to O6-benzylguanine (25 µmol/L) for 1 h before increasing doses of temozolomide (200–2,000 µmol/L). As a control, we measured the sensitivity of parental K562 cells (bullet) to temozolomide. K562 cells were plated at 1,000 per dish. After 10 to 12 d, colonies were enumerated.

 
Enrichment of P140K-MGMT–, MGMT-2–, and MGMT-5–Transduced K562 Cell Clones after Stringent Selection with O6-Benzylguanine Temozolomide or O6-Benzylguanine BCNU
To identify whether there was competitive survival and expansion of K562 clones expressing a particular O6-benzylguanine–resistant MGMT mutant and whether this depended on the drug used, we isolated K562 clones from methylcellulose and sequenced the mutant MGMT from sets of clones before and after drug treatment. As shown in Table 3 , a total of 38 clones were sequenced from K562 clones without selection; 55 clones were sequenced after temozolomide selection and 55 clones were sequenced after BCNU selection. In the O6-benzylguanine and temozolomide competitive selection studies, P140K-MGMT–transduced cells were enriched 1.7-fold and MGMT-2 was enriched 1.2-fold (P < 0.01, {chi}2 for the trend in distribution). In the O6-benzylguanine and BCNU competitive selection studies, cells transduced with MGMT-2 were enriched 1.5-fold, whereas MGMT-5 was enriched 1.4-fold (P = 0.18, {chi}2). K562 clones transduced with the two other O6-benzylguanine–resistant MGMT mutants (G156A-MGMT and MGMT-3) survived the drug treatment but were underrepresented among the clones isolated and sequenced. The rank order of O6-benzylguanine–resistant MGMT mutants selected under these enrichment conditions differed after temozolomide compared with BCNU selection. In addition, the selection pressure for temozolomide seemed greater than with BCNU because the range of enrichment between the O6-benzylguanine–resistant MGMT mutants was 4.9-fold with temozolomide but only 2.1-fold with BCNU. This may represent both intrinsic properties of drug selection and the dose intensity used.


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  		Table 3. Clonal O6-benzylguanine–resistant MGMT enrichment in K562 cells after drug treatment

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we compared the in vitro efficacy of five O6-benzylguanine–resistant MGMT mutants in protecting K562 cells against cytotoxicity induced by temozolomide and BCNU. We found that competitive, high-stringency drug selection identified the most potent drug-resistant MGMT mutant, and that this O6-benzylguanine–resistant MGMT mutant differed, depending on the drug used. The competitive in vitro selection and regrowth strategy is more discriminating than a simple survival assay and has been used to identify other drug resistance mutants, such as thymidine kinase (23), thymidylate synthase (24), dihydrofolate reductase (25), and glutathione S-transferase (26).

Our primary goal was to determine whether any of the five O6-benzylguanine–resistant MGMT mutants would be lost during selection, and whether we could actually select from among these remarkably O6-benzylguanine–resistant mutant proteins during high-dose O6-benzylguanine exposure and drug treatment. We hypothesized that G156A-MGMT might be lost because it is less O6-benzylguanine resistant, but it was not. Because all of the remaining four MGMT mutants are highly O6-benzylguanine resistant, we might not have observed any selection. Instead, we found that after selection with O6-benzylguanine and temozolomide, the most highly enriched mutant was P140K-MGMT, with an enrichment factor of 1.7 fold (P = 0.01). In contrast, resistance to O6-benzylguanine and BCNU resulted in enrichment in favor of MGMT-2 and MGMT-5 by 1.5- and 1.4-fold, respectively. This selection only reached P = 0.18; thus, the selection was quite modest compared with O6-benzylguanine and temozolomide. MGMT-2 and MGMT-5 also proved to be the most O6-benzylguanine–resistant MGMT mutants in our previous study, where 11 different MGMT multiple mutants (20), not including P140K-MGMT and G156A-MGMT, were competed, and the enrichment factor for MGMT2 and MGMT5 was 10- and 6-fold, respectively. This study confirms those results.

One factor that might skew the results is to have one of the O6-benzylguanine–resistant MGMT mutants transcribed or expressed at lower levels. This might overwhelm the ability to compete against another of the mutants. We analyzed this and found a similar distribution of gene expression and protein production among the mutants. On the other hand, the variation in viral production was not a factor because we analyzed the relative not the absolute enrichment after drug selection.

It is of interest that the P140K-MGMT mutant came in first with temozolomide and third during selection with O6-benzylguanine and BCNU. Intrinsic properties of the various O6-benzylguanine–resistant MGMT mutants likely contribute to the selection competition. The MGMT-3 mutant is the most stable of the mutant AGTs (19, 20). Stability and expression levels are important for repair of O6-methylguanine adducts generated by temozolomide because large numbers of adducts are formed, and removal of most of these adducts is required to protect the cell (27). In contrast, smaller numbers of BCNU-directed cross-links kill the cell; thus, a few highly O6-benzylguanine-resistant molecules of AGT are sufficient, whereas stability and total expression level are less important. The fact that the G156A-MGMT mutant, whereas not the most potent, was not lost during either competitive drug selection suggests that the degree of O6-benzylguanine resistance and protein stability are sufficient to repair DNA lesions in cells. This result is consistent with the ability of G156A-MGMT to protect CD34 cells in vitro (21), LTC-IC (28, 29), murine repopulating progenitor cells transplanted in vivo (30, 31), to increase the therapeutic efficacy of O6-benzylguanine and BCNU when transduced into host marrow in tumor xenograft models (32) and in protecting hematopoietic cells against O6-benzylguanine and temozolomide treatment in vitro (33). Although clinical concentrations of O6-benzylguanine observed in phase I trials are up to 20 µmol/L (4, 10, 34), this approach was taken in an attempt to identify whether strong selection favor to select AGT with multiple mutations compared with single amino acid substitution, such as G156A-MGMT, which shows to be moderately O6-benzylguanine resistant and the least stable AGT protein. In addition, among the other O6-benzylguanine–resistant MGMT mutants, which showed to be highly O6-benzylguanine resistant, we investigate whether some preferential amino acid substitutions would confer advantage to repair O6-methylguanine or O6-chloroethylguanine adducts on DNA. Surprisingly, G156A-MGMT was not lost during the competition study, and it shows the relative potency of G156A-MGMT in repair either O6-methylguanine or O6-chloroethylguanine lesions in DNA. Moreover, although P140K-MGMT was not preferred in the direct competition for O6-benzylguanine and BCNU resistance, it is a stable AGT protein (22) and has shown efficacy as an O6-benzylguanine and BCNU resistance gene protecting murine progenitor cells (35) in vitro and in vivo and in a dose-intensive tumor xenograft (36).

In summary, we have developed a direct competitive selection approach to analyze O6-benzylguanine–resistant MGMT mutant enrichment after selection with either O6-benzylguanine and temozolomide or O6-benzylguanine and BCNU. This approach can detect differences not seen in comparative clonal survival curves. We find that P140K-MGMT is preferred during O6-benzylguanine and temozolomide selection, whereas the multiple amino acid–substituted MGMT-2 is slightly (P = 0.18) preferentially enriched after O6-benzylguanine and BCNU. However, even the moderately O6-benzylguanine–resistant mutant G156A-MGMT is not lost. This suggests that MGMT-2, in particular, should be added to the list of effective O6-benzylguanine–resistant MGMT mutants that would be useful during drug resistance gene transfer into hematopoietic stem cells, and that the choice of O6-benzylguanine–resistant MGMT mutant should depend on the drug selection anticipated.


   Acknowledgments
 
We thank Jane Reese and Lili Liu for many helpful discussions.


   Footnotes
 
Grant support: USPHS grants RO1CA73062, RO1ES06288, UO1CA75525, and P30CA43703.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: A.M. Fontes is currently at the Regional Blood Center of Ribeirão Preto, Ribeirão Preto, SP, 14051-140 Brazil. B. Davis is currently at Virexis, Gaithersburg, Maryland. L. Encell is currently at Tanox, Inc., Department of Molecular Biology, 10301 Stella Link, Houston, TX 77025.

4 http://www.stanford.edu/group/nolan/protocols/pro_helper_dep.html Back

Received 7/11/05; revised 10/ 7/05; accepted 10/28/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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