Functional analysis of the involvement of apurinic/apyrimidinic endonuclease 1 in the resistance to melphalan in multiple myeloma
- Jiayin Xie†1Email author,
- Liang Zhang†1,
- Mengxia Li1,
- Jia Du1,
- Liwei Zhou1,
- Senlin Yang1,
- Linli Zeng1,
- Zengpeng Li2,
- Ge Wang1 and
- Dong Wang1
© Xie et al.; licensee BioMed Central Ltd. 2014
Received: 17 July 2013
Accepted: 26 December 2013
Published: 8 January 2014
Melphalan resistance has been considered one of the major obstacles to improve outcomes in multiple myeloma (MM) therapy; unfortunately, the mechanistic details of this resistance remain unclear. Melphalan is a highly effective alkylating agent which causes many types of DNA lesions, including DNA base alkylation damage that is repaired by base excision repair (BER). We postulated that human apurinic/apyrimidinic endonuclease 1 (APE1), an essential BER enzyme, plays a vital role in acquired melphalan resistance. However, because APE1 is a multifunctional protein with redox activity and acetylation modification in addition to its major repair activity, the particular APE1 function that may play a more important role in melphalan resistance is unknown.
Two MM cell lines, RPMI-8226 and U266 were used to measure the difference in APE1 levels in melphalan-resistant and sensitive derivatives. APE1 functional mutants for DNA repair, redox and acetylation were employed to investigate the roles of individual APE1 activities in acquired melphalan resistance.
Our results indicate that APE1 is overexpressed in both MM melphalan-resistant cells. Knocking down APE1 sensitizes the melphalan resistant MM cells to melphalan treatment. The exogenous expression of DNA repair mutant H309N and acetylation mutant K6R/K7R of APE1 failed to restore the melphalan resistance of the APE1 knockdown RPMI-8226 cells. The AP endonuclease activity and multidrug resistance protein 1 (MDR1) regulatory activity may play roles in the melphalan resistance of MM cells.
The present study has identified that the DNA repair functions and the acetylation modification of APE1 are involved in melphalan resistance of MM cells and has also shed light on future therapeutic strategies targeting specific APE1 functions by small molecule inhibitors.
KeywordsAcquired melphalan resistance Multiple myeloma Human apurinic/apyrimidinic endonuclease 1 DNA repair Base excision repair
Primary drug resistance in multiple myeloma (MM) patients has created a hurdle to consistently successful chemotherapeutic outcomes. Despite gradual advances in treatment using optimized strategies that combine multiple agents, effective remission is achieved in a sub-optimal number of patients (fewer than 50% of patients) . The current standard of care for elderly MM patients includes the nitrogen mustard alkylating agent melphalan in conjunction with prednisone. Melphalan primarily distorts the DNA guanine base with an alkyl group monoadduct , particularly at the nitrogen atom 7 of the imidazole ring, and it can also distort DNA with other adducts. Several suspected means of in vitro resistance to these mustard agents include cytokine production defects in the bone marrow milieu, altered drug delivery by transporters that effectively decrease cellular drug absorption, and an increase in effective DNA repair of mustard-specific lesions [3, 4]. Currently it is unclear which of these pathways contributes to drug resistance during chemotherapeutic regimens.
Among all the previously-proposed mechanistic models, enhanced DNA repair capacity has been closely associated to melphalan resistance in MM patients [5, 6]. DNA repair function has been widely accepted as the most important mechanism of resistance to anticancer drugs, especially those specifically targeting DNA. Although there are 5–6 major DNA repair pathways, the major type of DNA damage caused by melphalan is base alkylation which is mainly repaired by DNA base excision repair (BER) mechanisms . Melphalan-induced alkylated bases are recognized and removed by particular glycosylases such as methylpurine glycosylase (MPG) leaving an abasic site with the N-glycosyl bond intact. Then human apurinic/apyrimidinic endonuclease 1 (APE1) cleaves the DNA backbone at the abasic site, leaving an exposed 3′OH and 5′ deoxyribose phosphate. Following the function of APE1, DNA polymerase beta incorporates the correct nucleotide followed by nick ligation by ligase III. In the BER pathway, the activity of APE1 largely determines the effectiveness of this DNA repair . APE1 is an essential protein for many cellular processes  and its biological importance is highlighted by early embryonic lethality in mouse Apex1, the homologue of human APE1, knockout mice . A number of preclinical functional studies revealed that APE1 is more highly expressed in various types of tumor tissues which supposedly contributes to cancer cell survival and proliferation [11, 12]. Moreover, overexpression of APE1 in tumor tissues is usually closely correlated with a less effective response or resistance to cancer therapeutic agents [13, 14]. Although the role of APE1 in drug resistance has been established and widely accepted, the detailed mechanisms for individual therapeutic agents may vary and are not yet fully understood.
We previously reported that APE1 is involved in melphalan resistance in the MM cell line KM3 by using a tissue-specific inducible APE1 silencing vector . As a result, we confirmed that APE1 is a promising therapeutic target and that suppressing the expression of APE1 might enhance melphalan treatment in MM patients. However, APE1 is a multifunctional protein with at least two distinct activities which play different roles in drug resistance. As mentioned above, APE1 is the essential enzyme of DNA base excision repair . On the other hand, APE1 is a redox factor regulating important agents involved in oxidative stress, including NF-κB, AP-1, p53, and Egr-1 . Notably, recent studies indicate a novel role of APE1 in regulating MDR1 expression through an acetylation-dependent mechanism . The membrane-associated protein encoded by the Mdr-1 gene is a member of the superfamily of ATP-binding cassette (ABC) transporters which functions as an ATP-dependent drug efflux pump for xenobiotic compounds . Therefore, it is responsible for decreased drug accumulation in multidrug-resistant cells which further facilitates the development of drug resistance.
Taken together, overexpression of APE1 in MM cells promotes resistance to melphalan, possibly through different mechanisms, while the involvement of different activities of APE1 in this process remains unknown. Therefore, we initiated the present study to explore which APE1 functions are involved in melphalan resistance in MM cells. We utilized APE1 overexpression or RNAi vectors to measure the impact of manipulating APE1 on melphalan resistance of the MM cell lines. Additionally, we used APE1 function-specific or post-transcriptional modification site mutant overexpression vectors to differentiate the impact of specific APE1 activities on melphalan resistance. Our results indicated that APE1 overexpression and manipulation in melphalan resistant MM cells affects melphalan resistance. The DNA repair activity and MDR1 regulatory activity of APE1 are mainly involved in the melphalan resistance of RPMI-8226 MM cells while DNA repair activity is more important in cell survival following melphalan treatment.
Cell and reagents
RPMI 1640 medium, Opti-MEM® I Reduced-Serum Medium and fetal bovine serum, Lipofectamine™ 2000 Transfection Reagent, TRIzol RNA isolation reagent and primers were from Invitrogen (Grand Island, NY). Cell Counting Kit-8 (CCK-8), melphalan, myrecitin, synthetic siRNA against APE1 and MDR1 were from Sigma-Aldrich (St Louis, MO). Tetrahydrofuran-containing oligonucleotides, biotin-conjugated oligos and all other regular oligos were synthesized from Takara (Dalian, China). T4 polynucleotide kinase (T4 PNK), T4 ligase, restriction endonucleases, and high-fidelity Pfu DNA polymerase were from Promega (Madison, WI). Halt protease inhibitor cocktail, LightShift chemiluminescent EMSA kit, Super Signal West Pico chemiluminescent reagents, horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies were from Pierce (Rockford, IL). The chemo-sensitive RPMI-8226 and U266 cell line were purchased from American Type Culture Collection (Manassas, VA) and their melphalan-resistant sublines RPMI-8226/LR5 and U266/LR6 were obtained from Dr. William S. Dalton (Lee Moffitt Cancer Center, Tampa, FL). All MM cell lines were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin (Hyclone, Logan, UT), and maintained at 37°C in a humidified atmosphere in the presence of 5% CO2-95% air.
Constructs and transfection
The constructs of APE1 knockdown and wildtype or overexpression mutants used in this study included pTer/APE1 (shAPE1), p3XFLAG-CMV/APE1WT (APE1WT), p3XFLAG-CMV/APE1H309N (APE1H309N), p3XFLAG-CMV/APE1C65S (APE1C65S), and p3XFLAG-CMV/APE1K6R/K7R (APE1 K6R/K7R), kind gifts from Dr. Gianluca Tell (University of Udine, Udine, Italy). The APE1 eukaryotic overexpression vector was constructed based on the pcDNA-3.1 vector. The detailed procedures were reported previously [9, 20]. The transfections were performed using Lipofectamine™ 2000 Transfection Reagent following the manufacturer’s protocol for transient transfection of suspension cells.
Cells (2 × 105) on 6-well plates were transfected or treated as indicated. Cell viability was evaluated by MTT assay at various time points after transfection or treatment. Cell-counting kit-8 reagent was added to each dish at a concentration of 1/10 volume, and the plates were incubated at 37°C for an additional 4 h. Absorbance was then measured at 490 nm and at 630 nm as a reference with a Microplate Reader 550 (Bio-Rad Laboratories, Hercules, CA). Cell viability (%) = OD value of treatment group/OD value of control group × 100%.
Western blot and antibodies
Western blots were performed as previously described . Suppliers and incubation conditions of antibodies used for Western blots were as follows: anti-APE1 monoclonal (Novus), 1 h at 37°C, dilution 1:5000; anti-MDR1 monoclonal (Sigma), dilution 1:500; HRP-conjugated anti-acetylation lysine antibody (Milipore), dilution 1:1000, overnight at 4°C; anti-β-actin monoclonal (Sigma), 1 h at 37°C, dilution 1:2000.
Expression of the APE1 gene was detected by real-time RT-PCR and normalized by control gene β-actin expression. Total RNA was extracted using the TRIZOL reagent and then reverse-transcribed into single-stranded DNA using PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). Real-time RT-PCR was performed with a Lightcycler 480 real-time RT-PCR system (Roche Diagnostics). APE1 forward primer, 5′-TGGAATGTGGATGGGCTTCGAGCC-3′ and APE1 reverse primer, 5′-AAGGAGCTGACCAGTATTGATGA-3′ were utilized.
Oligonucleotide cleavage assay
The AP endonuclease activity of APE1 was evaluated by a well-characterized oligonucleotide cleavage assay . Briefly, a 51-mer oligonucleotide containing a THF site, the analogue of an abasic site, at the 22nd position was 5′-end radiolabeled. The labeling reaction consisted of 10 pmol of the single stranded oligonucleotide, 2.5 pmol of γ32P-ATP, T4 PNK, and appropriate kinase buffer in a total volume of 10 μl. Reactants were incubated for 30 minutes at 37°C and 5 minutes at 95°C. Complementary oligonucleotide was then added and cooled down to 22°C to form duplex DNA. Activity assays contained 0.5 pmol of labeled duplex oligonucleotide, 1 × REC Buffer [50 mM HEPES, 50 mM KCl, 10 mM MgCl2, 1% (w/v) bovine serum albumin, 0.05% (v/v) Triton X-100 (pH 7.5)], protein extraction (0 to 10 μg) in a 10 μl reaction volume and were incubated at 37°C for 15minutes. The reactions were terminated by adding 10 μl formamide with dyes. Equal volumes (20 μL) of the reaction products from assays of AP endonuclease activity were resolved in a 20% polyacrylamide gel with 7 M urea in 1 × Tris-borate EDTA buffer at 300 V for 40 minutes. Wet gels were autoradiographed at −70°C overnight.
Electrophoretic mobility shift assay (EMSA)
EMSA was performed according to the LightShift Chemiluminescent EMSA kit user’s instructions with minor modifications. Briefly, 5 μg of nuclear extracts were incubated with 3′-biotin labeled, purified double-stranded oligonucleotide probes. The probes containing NF-κB consensus sites: NF-κBF: 5′-AGTTGAGGGGACTTTCCCAGGC-3′ and NF-κBR: 5′- CGGACCCTTTCAGGGGAGTTGA -3'were synthesized and labeled with biotin at the 3′ end. After incubation for 30 min at room temperature, samples were separated on a pre-run 5% polyacrylamide gel at 100 V for 90 min and then transferred to Zeta-Probe GT nylon membrane (Bio-Rad). The probes were detected by HRP-conjugated streptavidin (1:300) and the bands were visualized by ECL reagents provided with the kit. The resultant bands were quantified using the imaging software Quantity One (Bio-Rad).
The cells were rinsed twice with ice-cold PBS and harvested, and the cell suspension was exposed to melphalan on ice for 15 min. Immediately after treatment or after a 30 min recovery incubation at 37°C post treatment, the cell suspension was stored on ice and an alkaline comet assay performed using a Comet assay kit (Trevigen, USA) according to the manufacturer’s instructions with modifications.
Cells were harvested by scraping and washed once with ice-cold phosphate-buffered saline (PBS). Cell pellets were resuspended and incubated in IP lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China) supplemented with protease inhibitor cocktail (Pierce) at a cell density of 107 cells/ml on ice for 30 min. After centrifugation at 12,000 × g for 10 min at 4°C, the supernatant was collected as total cell lysate. Protein concentration was determined by using the Bradford assay (Bio-Rad, Hercules, CA). Samples were pre-cleared by incubating with protein A/G agarose resin for 30 min on ice then coimmunoprecipitated for 3 h using APE1 antibody (Novus) following the manufacturer’s instructions. After incubation, protein A/G agarose resin was then added and incubated for 1 hour at 4°C. After washing 3 times with PBS containing protease inhibitor, the pellet containing agarose beads together with binding proteins were mixed with sample buffer and incubated at 100°C for 5 min. The samples were then stored at −80°C or subjected to Western blotting analysis immediately.
The differential expression of APE1 in RPMI-8226 and U266 parental cell and their drug-resistant cell lines RPMI-8226/LR5 and U266/LR6
Manipulation of APE1 affects cell resistance to melphalan
DNA repair is involved in melphalan-resistant MM cells
The DNA repair activity of APE1 plays an important role in melphalan resistance
MDR1 expression is reduced in APE1 deficient MM cells
Discussion and conclusion
Despite the adverse side effects caused by alkylating agents on bone marrow and other normal tissues, melphalan remains one of the most commonly prescribed chemotherapies in MM patients. As the main treatment regimen of MM, melphalan greatly affects the outcome of overall treatment through its therapeutic efficacy. However, a considerable variation of therapeutic response to melphalan is observed clinically. We previously found that melphalan cytotoxicity closely correlates to the expression level of the multifunctional gene APE1 . Our present results indicate that APE1 has a higher expression level in the melphalan resistant cell line RPMI-8226/LR5 and that expression of APE1 is effectively induced by melphalan in a dose- and time-dependent manner. We further employed APE1 knockdown and overexpression vectors to exogenously manipulate the APE1 levels in RPMI-8226/LR5 and its parental cell line RMPI-8226 which affected melphalan cytotoxicity. These results further reinforced the critical regulatory role of APE1 in melphalan resistance in different MM cell line models in accordance with our previous observations. Subsequently, we determined which functions of APE1 were critical for melphalan resistance by taking advantage of developed melphalan-resistant MM cell lines and APE1 function-specific mutant expression vectors. By comparing the different capacities of restoring resistance to APE1-knockdown RPMI-8226 cells by three APE1 functional mutants, the DNA repair activity and the intact acetylation residues K6 and K7 were shown to play critical roles in the development of melphalan resistance of MM cells. Then we performed mechanistic studies of DNA repair activity and acetylation modification of APE1 and the impact on melphalan cytotoxicity. Altogether our results identify the most important functions of APE1 in melphalan resistance of MM cells and shed light on future therapeutic strategies targeting specific APE1 functions by small molecule inhibitors.
As predicted, the DNA repair activity of APE1 plays the most important part in melphalan resistance. Melphalan, as a typical alkylation agent, exerts its cancer cell killing effect through DNA damage. Although the most cytotoxic lesion caused by melphalan is considered to be the interstrand crosslinks, the majority of DNA lesions are N7G monoadducts (~38%) and N3A monoadducts (~20%)  which are also potentially lethal by blocking DNA replication. DNA base alkylation and oxidative lesions are mainly repaired by BER, so it is plausible that the abasic site endonuclease activity of APE1 functions as a major mechanism in acquired melphalan resistance. Although the various single base lesions require different lesion-specific glycosylases, APE1 is common to BER and incises the backbone of the DNA strand and facilitates gap filling by DNA Polβ. Therefore, the activity of APE1, essential for BER, determines the repair capacity of mono methylation induced by alkylation agents including MMS  and melphalan. In addition, DNA repair activity is associated with the sensitivity of different agents targeting DNA which mainly cause different types of lesions including cisplatin , 5-FU , bleomycin, and ionizing radiation (IR) . Although the detailed mechanisms of the repair activity of APE1 in drug resistance remain unclear, these observations imply a more important role of APE1 in promoting cell survival though a DNA repair-dependent mechanism.
We recently observed that APE1 was highly expressed in bone marrow stromal cells (BMSCs) in MM patients compared to the normal donors. This study provided a plausible explanation to the drug-resistance of MM by APE1 in that APE1 regulates cytokines, including IL-6 and IL-8, produced in BMSCs through redox regulation of NF-κB and AP-1 . This study merely focused on the role of the microenvironment of MM. However, our present results indicate that the redox activity of APE1 is not involved in acquired melphalan resistance as we expected. We actually observed the reduction of IL-6/8 mRNA in redox activity deficient cells (data not shown), and the reduction of IL-6/8 expression has a minor impact on cell survival after melphalan treatment. The paracrine agents from the BMSCs in the microenvironment of the bone marrow are the major source of cytokines and growth factors for MM cell survival; hence, it is probable that the autocrine cytokines from MM cells have little effect on drug resistance.
Interestingly, we found that APE1 regulates the sensitivity of MM cells to melphalan by affecting MDR1 expression. This MDR1 regulatory role of APE1 is exclusively dependent on the integrity of acetylation sites at K6/K7 as reported previously . A previous study indicated that the MDR1 expression level was associated with low intracellular accumulation and low cytotoxicity of melphalan in different hematopoietic cancer cell lines, including seven MM cell lines . In accord with our study, the MDR1 inhibitor successfully reversed melphalan resistance in MDR1 overexpressed HL-60 cells. However, the regulatory role of APE1 in melphalan sensitivity occurs only partially through MDR1 expression. As shown in Figure 6, knockdown of MDR1 in APE1-overexpressed RPMI-8226 cells only partially restores sensitivity to melphalan compared to the MDR1 knockdown in RPMI-8226 cells.
In this present study we are the first to identify, to our knowledge, that the APE1 DNA repair function, together with acetylation modification, plays the most important role in melphalan resistance. Although we demonstrated the critical function of APE1 in melphalan resistance through cell models in this current project, the detailed molecular mechanisms in intrinsic resistance to melphalan are still unknown. Since different mechanisms may be involved in intrinsic and acquired melphalan resistance, more work needs to be done using different cell models to determine the different functions of APE1 in intrinsic and acquired melphalan resistance.
Base excision repair
Bone marrow stromal cells
Cell counting kit-8
Electrophoretic mobility shift assay
Multidrug resistance protein 1
This work is supported by National Natural Science Foundation of China (No. 30772520 and No. 81172258) to Prof. Jiayin.Xie. The authors are grateful to Dr. William S. Dalton for the generous gift of RPMI-8226/LR5 and to Dr. Gianluca Tell for APE1 shRNA, wildtype cDNA, and functional mutation expression vectors.
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