ONCOLOGY LETTERS 10: 845-849, 2015 Leukemia cells are sensitized to temozolomide, carmustine and melphalan by the inhibition of O6‑methylguanine‑DNA methyltransferase HAJIME ARAI, TAKAHIRO YAMAUCHI, KANAKO UZUI and TAKANORI UEDA Department of Hematology and Oncology, Faculty of Medical Sciences, University of Fukui, Eiheiji, Fukui 910-1193, Japan Received August 8, 2014; Accepted April 13, 2015 DOI: 10.3892/ol.2015.3307 Abstract. The cytotoxicity of the monofunctional alkylator, Introduction temozolomide (TMZ), is known to be mediated by mismatch repair (MMR) triggered by O6-alkylguanine. By contrast, the Alkylating agents comprise a major class of chemo- cytotoxicity of bifunctional alkylators, including carmustine therapeutic agents, widely used in various types of cancer, (BCNU) and melphalan (MEL), depends on interstrand cross- including leukemia (1,2). There are two types of alkyl- links formed through O6-alkylguanine, which is repaired by ating agents: monofunctional and bifunctional agents. nucleotide excision repair and recombination. O6-alkylguanine Bifunctional alkylating agents include cyclophosphamide, is removed by O6-methylguanine-DNA methyltransferase ifosfamide, melphalan (MEL) and carmustine (BCNU; (MGMT). The aim of the present study was to evaluate the cyto- also known as 1,3-bis(2-chloroethyl)-1-nitrosourea). toxicity of TMZ, BCNU and MEL in two different leukemic Monofunctional agents include temozolomide [TMZ; cell lines (HL-60 and MOLT-4) in the context of DNA repair. also known as 3,4-dihydro-3-methyl-4-oxoimidazo The transcript levels of MGMT, ERCC1, hMLH1 and hMSH2 (5,1-d)-as-tetrazine-8-carboxamide] and dacarbazine (1-3). were determined using reverse transcription-quantitative Alkylating agents form a variety of DNA adducts in polymerase chain reaction. In addition, the proliferation was cancer cells, including mono-adducts on N1-alkylguanine, measured using the trypan blue exclusion assay. Drug sensi- N3-alkyladenine, N7-alkylguanine or O6-alkylguanine, and tivity was found to vary between the two cell lines. Treatment di-adducts between or within DNA strands (1-4). Bifunctional of the cells with TMZ, BCNU or MEL in combination with alkylating agents result in cytotoxicity due to the production O6-benzylguanine, an MGMT inhibitor, was demonstrated of interstrand crosslinks, which are formed through the to sensitize the two cell lines to these agents. However, the intermediate production of O6-alkylguanine (5,6). These extent of sensitization was not found to be correlated with the crosslinks are repaired through nucleotide excision repair expression levels of MGMT transcripts. Furthermore, the drug (NER) and recombination. By contrast, monofunctional sensitivity was also not associated with the transcript levels agents generate persistent O6-methylguanine adducts that of ERCC1, hMLH1 and hMSH2. Thus, leukemic cells were initiate futile cycling of the DNA mismatch repair (MMR) sensitized to alkylating agents by the inhibition of MGMT. pathway, which causes DNA double-strand breaks (7-10). Intact MMR is required for the exertion of the cytotox- icity of monofunctional agents. The DNA repair enzyme, O6-methylguanine-DNA methyltransferase (MGMT), repairs O6-alkylguanine adducts (11,12) and reverses the cytotoxicity induced by the two types of alkylating agents. Correspondence to: Dr Takahiro Yamauchi, Department of The cytotoxic effects of alkylating agents are limited Hematology and Oncology, Faculty of Medical Sciences, University by a number of factors, including DNA repair (2,13,14). In of Fukui, 23 Shimoaizuki, Matsuoka, Eiheiji, Fukui 910-1193, Japan the present study, the cytotoxic effects of the bifunctional E-mail: [email protected] BCNU and MEL agents, as well as the monofunctional TMZ agent, were evaluated in relation to DNA repair. The Abbreviations: IC50, 50% growth-inhibitory concentration; MMR, mismatch repair; MGMT, O6-methylguanine-DNA methyltransferase; effects were compared in two cultured leukemia cell lines, NER, nucleotide excision repair; BG, O6-benzylguanine; HL-60 and MOLT-4. In addition, the sensitivity of the cells TMZ, temozolomide or 3,4-dihydro-3-methyl-4-oxoimidazo was manipulated by the addition of an MGMT inhibitor, 6 (5,1-d)-as-tetrazine-8-carboxamide; BCNU, carmustine or O -benzylguanine (BG). The extent of the drug cytotox- 1,3-bis(2-chloroethyl)-1-nitrosourea; MEL, melphalan icity was analyzed to determine its correlation with DNA repair, including any associations with MGMT, NER and Key words: DNA repair, temozolomide, carmustine, melphalan, MMR (15,16). 6 O -benzylguanine Our previous study demonstrated the important role of MMR in the exertion of the cytotoxicity of monofunctional agent temozolomide (17). Restored MMR sensitized the 846 ARAI et al: SENSITIZATION OF LEUKEMIC CELLS TO ALKYLATING AGENTS cancer cells to temozolomide. Therefore, the aim of the Table I. Drug sensitivities of the two leukemia cell lines. present study was to evaluate the cytotoxicity of alkylating agents from the viewpoint of MGMT. IC50 (µM) ------------------------------------------------------------- Drugs HL-60 MOLT-4 Materials and methods TMZ 49.0 191.5 Chemicals and reagents. BCNU, MEL and BG (all BCNU 10.0 22.0 purchased from Sigma-Aldrich, St. Louis, MO, USA) were dissolved in 99% ethanol immediately prior to use. TMZ MEL 4.5 1.5 (Schering-Plough KK, Osaka, Japan) was dissolved in 100% TMZ+BG 4.5 169.0 dimethyl sulfoxide immediately prior to use. BCNU+BG 3.0 1.4 MEL+BG 0.3 1.4 Cell culture. Human acute myeloid leukemia cell line, HL-60, and human acute T lymphoblastic leukemia cell line, Cells were incubated with various concentrations of TMZ, BCNU MOLT-4, were used in this study (JCRB Cell Bank, Osaka, or MEL, with or without BG. The IC50 values were then determined using the trypan blue exclusion assay. TMZ, temozolomide; BCNU, Japan). The cells were cultured in RPMI 1640 medium (Life 6 carmustine; MEL, melphalan; BG, O -benzylguanine; IC50, 50% Technologies Japan, Ltd., Tokyo, Japan) in a humidified growth-inhibitory concentration. atmosphere with 5% CO2 at 37˚C. Drug treatment and proliferation assay. To evaluate the growth-inhibitory effect of each agent on the two cell lines, Table II. Sensitization by the addition of BG. the trypan blue exclusion assay was performed (17,18). Briefly, the cells were incubated with various concentra- Ratio of IC50 tions of TMZ, BCNU or MEL (10 nM, 100 nM, 1 µM or ----------------------------------------------------- 10 µM), alone or in combination with BG (10 µM), for 72 h. Drugs HL-60 MOLT-4 Subsequently, the samples were stained with trypan blue TMZ / TMZ+BG 10.9 1.1 (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and the viable cells, which exhibited negative staining, were BCNU / BCNU+BG 3.3 15.7 MEL / MEL+BG 15.0 1.1 counted. The 50% growth-inhibitory concentration (IC50) was the concentration at which 50% of the growth of the Ratios of IC values = (IC value of TMZ, BCNU or MEL treat- untreated cells was inhibited. This value was extrapolated 50 50 ment) / (IC50 value of TMZ, BCNU or MEL treatment in combination from the growth curve drawn for each drug treatment, with with BG). TMZ, temozolomide; BCNU, carmustine; MEL, melphalan; 6 100% considered to be the condition of untreated cells. BG, O -benzylguanine; IC50, 50% growth-inhibitory concentration. Reverse transcription‑quantitative polymerase chain reac‑ tion (RT‑qPCR). The transcript levels of MGMT, ERCC1, hMLH1 and hMSH2 were determined by RT-qPCR using was used for determination of any correlation between two the ABI Prism 7700 sequence detection system (Applied parameters. All statistical analyses were performed using Biosystems Life Technologies, Foster City, CA, USA). Microsoft Excel 2007 (Microsoft Corporation, Redmond, RT-qPCR was performed according to the method of our WA, USA). P<0.05 was considered to indicate a statistically previous study (17). ERCC1 is responsible for incision significant difference. of the damaged DNA strand in the NER pathway, while hMLH1 and hMSH2 are two key factors in the MMR Results response. For MGMT, the sense primer sequence was 5'-TCCCGTTTTCCAGCAAGAGTC-3', and the antisense Growth‑inhibitory effects of the alkylating agents. The sequence was 5'-GGGCTGCTAATTGCTGGTAAGA-3'. growth-inhibitory effects of TMZ, BCNU and MEL were The TaqMan probe DNA sequence was FAM-CCAGACA evaluated in the HL-60 and MOLT-4 cells. Overall, the sensi- GGTGTTATGGAAGCTGCTGAAG-TAMRA (Mitsubishi tivity of the two cell lines to these agents varied; however, Kagaku Bio-Clinical Laboratories, Tokyo, Japan). In addition, treatment with MEL appeared to be the most effective in the primers for ERCC1, hMLH1 and hMSH2 were purchased inhibiting the cell growth (Table I). from Mitsubishi Kagaku Bio-Clinical Laboratories. The absolute standard curve quantitation method was used for Inhibition of MGMT by BG. The cytotoxic effect of alkyl- MGMT and ERCC1, and the relative standard curve quanti- ating agents is generally reduced by DNA repair in cancer tation method was used for hMLH1 and hMSH2. The values cells (3-6). Upon treatment of the HL-60 and MOLT-4 cells of HL-60 cells were set to 1 and relative values were deter- with TMZ, BCNU or MEL in the presence of BG, an MGMT mined for the MOLT-4 cells. inhibitor, the two cell lines were apparently sensitized to all these agents (Table I). However, the extent of sensitization Statistical analyses. Graphs were generated using the varied among the drugs in the two cell lines (Table II; Fig. 1). GraphPad Prism software (version 5.0; GraphPad Software, BG was not found to be cytotoxic to cells in the previous Inc., San Diego, CA, USA). Spearman's rank correlation study (17). ONCOLOGY LETTERS 10: 845-849, 2015 847 A B C Figure 1. Changes in the IC50 values following the addition of BG. The cellular sensitivity to (A) TMZ, (B) BCNU or (C) MEL were determined in the HL-60 and MOLT-4 cells in the presence or absence of BG using the trypan blue exclusion assay. Values are expressed as the mean of at least three independent 6 experiments.