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Cancer Research 67, 5831-5839, June 15, 2007. doi: 10.1158/0008-5472.CAN-06-4533
© 2007 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Effect of a Single Nucleotide Polymorphism in the Murine Double Minute 2 Promoter (SNP309) on the Sensitivity to Topoisomerase II–Targeting Drugs

Mamatha S. Nayak, Jin-Ming Yang and William N. Hait

Departments of Pharmacology and Medicine, The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey/Robert Wood Johnson Medical School, New Brunswick, New Jersey

Requests for reprints: Jin-Ming Yang and William N. Hait, The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey/Robert Wood Johnson Medical School, 195 Little Albany Street, New Brunswick, NJ 08901. Phone: 732-235-8075; Fax: 732-235-8094; E-mail: jyang{at}umdnj.edu and haitwn{at}umdnj.edu.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A single nucleotide polymorphism (SNP) SNP309 (T->G) in the murine double minute 2 (MDM2) promoter creates a high-affinity Sp1 binding site and increases the expression of MDM2 mRNA and protein. Approximately 40% of the populations harbor at least one variant allele and 12% to 17% are homozygous G/G at codon 309. This MDM2 SNP increases susceptibility to cancer and decreases the response of cancer cells to certain forms of treatment, such as radiation therapy and DNA-damaging drugs. Topoisomerase II (TopoII)–targeting agents are commonly used chemotherapeutic drugs with a broad spectrum of activity. However, resistance to TopoII poisons limits their effectiveness. We show that MDM2 SNP309 rendered a panel of cancer cell lines that are homozygous for SNP309 selectively resistant (~10-fold) to certain TopoII-targeting chemotherapeutic drugs (etoposide, mitoxantrone, amsacrine, and ellipticine). The mechanism underlying this observation was Mdm2-mediated down-regulation of TopoII; on drug exposure, MDM2 bound to TopoII and resulted in decreased cellular enzyme content. Knockdown of MDM2 by RNA interference stabilized TopoII{alpha} and decreased resistance to TopoII-targeting drugs. Thus, MDM2 SNP309 (T->G) may represent a relatively common, previously unappreciated determinant of drug sensitivity. Given the frequency of SNP309 in the general population (40% in heterozygous T/G and 12% in homozygous G/G condition), our observation may have important implications for the individualization of cancer chemotherapy. [Cancer Res 2007;67(12):5831–9]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The status of the tumor suppressor protein, p53, affects the progression of many cancers as well as the effectiveness of chemotherapeutic drugs. Alterations in the p53 gene occur in >50% of human malignancies, and cancer cells with mutant p53 protein are less sensitive to treatment with most DNA-damaging agents than those with the wild-type (WT) protein (1–3). The murine double minute 2 (MDM2) gene encodes a protein that is a key component in the p53 signaling pathway. MDM2 is transcriptionally activated by p53 (4) and regulates its content by targeting p53 for ubiquitin-mediated degradation. Knockout of MDM2 in mice is embryonic lethal (5). However, knockout of both p53 and MDM2 allows embryos to survive, suggesting that the primary function of MDM2 is to regulate the actions of p53. MDM2 regulates the function of p53 in several ways. For example, it is the ubiquitin E3 ligase for p53 and targets its degradation by the proteasomal pathway (6); MDM2 is responsible for the nuclear to cytoplasmic shuttling of p53, thus inhibiting its function as a transcription factor (7). MDM2 also binds p53 and inhibits transactivation (8). Although mutations in MDM2 are rare, MDM2 protein is overexpressed in ~5% to 10% of human tumors (9). MDM2 overexpression can occur through gene amplification or through transcriptional or post-transcriptional mechanisms. MDM2 gene amplification has been observed in more than one third of human sarcomas (10, 11).

MDM2 also controls the activity of several proteins involved in cell cycle progression independently of p53. For example, MDM2 promotes the degradation of the phosphorylated retinoblastoma protein (pRB; ref. 12) and p21 (13) and regulates their activities. In addition, MDM2 interacts with the S phase-promoting factor, E2F1, and increases its function (14). Because of its ability to determine the fate of important regulators of the cell cycle, MDM2 might play several roles in determining the sensitivity of cancer to chemotherapeutic agents. For example, Kondo et al. (15) showed that overexpression of MDM2 resulted in expression of the MDR1 gene product, P-glycoprotein (P-gp), in human glioblastoma cells, resulting in decreased sensitivity to etoposide (VP-16) and doxorubicin. In contrast, McKenzie et al. (16) reported that MDM2 did not influence the ability of p53 to mediate sensitivity to DNA-damaging agents, such as doxorubicin and actinomycin D, in rhabdomyosarcoma cell lines engineered to overexpress p53.

Topoisomerase II{alpha} (TopoII{alpha}) is a nuclear enzyme that plays an important role in DNA topology and repair following DNA damage. Chen et al. (17) showed that TopoII{alpha} is the target of several antineoplastic drugs and that down-regulation of TopoII{alpha} results in resistance to several TopoII-targeting drugs, including VP-16. It has been reported previously that VP-16 forms a noncleavable complex with TopoII{alpha} and this complex is subject to ubiquitin-mediated degradation (18). Degradation of TopoII{alpha} also occurs during apoptosis induced by the adenovirus E1A protein (19). However, the mechanism that facilitates ubiquitin-mediated degradation of TopoII{alpha} remains unknown.

Recently, Bond et al. (20) discovered a single nucleotide polymorphism (SNP) in the MDM2 promoter that resulted in overexpression of MDM2 and attenuation of p53 function. This SNP (T->G change) is at the 309th nucleotide in the first intron of the MDM2 gene and results in overexpression of MDM2 RNA and protein. SNP309 occurs at a high frequency (40% heterozygous T/G and 12% homozygous G/G) in the general population. In both hereditary (Li-Fraumeni) and certain sporadic forms of cancers, the presence of SNP309 resulted in earlier age of onset of cancer and greater number of cancers than patients without this variant (20). Cell lines homozygous for SNP309 had an altered apoptotic response to VP-16 and that was attributable, at least in part, to the attenuated action of p53.

Therefore, we investigated the effect of MDM2 SNP309 on TopoII-targeting drugs. We observed that cancer cell lines harboring MDM2 SNP309 were resistant to certain TopoII-targeting drugs and that this could be attributable to changes in the rate of degradation of TopoII{alpha}.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines. The cancer cell lines, A875, T47D, Manca, CCF STTG1, Tera 2, ML1, H460, Saos 2, and WT mouse embryo fibroblasts (MEFs), p53 knockout MEFs, and p53 and MDM2 double knockout MEFs were generous gifts from Dr. Arnold Levine (The Cancer Institute of New Jersey, Brunswick, NJ). A875, Tera 2, and Saos 2 cells and MEFs were cultured in DMEM (Life Technologies) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Life Technologies). T47D, Manca, CCF STTG1, ML1, and H460 were cultured in RPMI 1640 (Life Technologies) supplemented with 10% FBS and 1% penicillin-streptomycin.

Drugs, reagents, and antibodies. VP-16 (Sigma Chemical Co.) was dissolved in ethanol (final concentration of 1%). Cisplatin, camptothecin, amsacrine, ellipticine, and mitoxantrone (Sigma Chemical) were dissolved in DMSO. [3H]VP-16 (250 µCi) was obtained from Moravek Biochemicals. Primary antibodies used for Western blotting were as follows: TopoII{alpha} Ab-1 (Labvision), MDM2 SMP14 (Santa Cruz Biotechnology), and p53 and monoclonal ß-actin clone AC15 (Sigma Chemical). The reactive proteins were visualized using enhanced chemiluminescence (ECL) detection (Amersham Pharmacia Biotech). Antibodies used for immunoprecipitation were as follows: TopoII AB (Topogen) and MDM2 SMP14. siGENOME SMARTpool MDM2 and siCONTROL nontargeting small interfering RNA (siRNA) 1 (Dharmacon) were transfected using Oligofectamine reagent (Invitrogen).

Cell proliferation assay. Cells indicated were plated in 96-well tissue culture plates, allowed to attach overnight, and then treated with increasing doses of VP-16, camptothecin, cisplatin, mitoxantrone, amsacrine, or ellipticine for 48 h. Twenty-five microliters of 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in PBS were then added to each well, and cells were incubated at 37°C for 4 h. Formazan crystals were dissolved in DMSO. Absorbances were determined at 570 nm using a Dynatech MR5000 plate reader. Viability was expressed as a percentage of control by dividing the absorbance of each treated sample by the average of the untreated controls. All the experiments were repeated four times and in triplicates. IC50 was defined as the concentration of drug that decreased cell viability by 50%.

Immunoblot analysis. Cell lines were plated at 2 x 106 cells in 60-mm dishes. After the respective treatments, cells were harvested and lysed in lysis buffer containing 50 mmol/L Tris-HCl (pH 7.5), 1% sodium deoxycholate, 1% Triton X-100, 150 mmol/L NaCl, 10 µg/mL leupeptin, 10 µg/mL pepstatin, 20 µg/mL aprotinin, 10 mmol/L sodium PPi, 50 mmol/L sodium fluoride, and 500 µmol/L sodium orthovanadate or in alkaline lysis buffer as described previously. The lysates were pulsed four to six times with 2.5 W using a VirSonic 60 VirTis Sonicator. Protein concentrations were determined by the method of Bradford using the Bio-Rad protein assay reagent (Bio-Rad). Fifty micrograms of protein were loaded onto 6% SDS-PAGE gels followed by transfer to nitrocellulose membranes (Schleicher & Schuell). The blots were assayed for the expression of TopoII{alpha}, MDM2, and p53 and enhanced by chemiluminescence detection (ECL). ß-Actin served as loading control.

Reverse transcription-PCR. Cells were plated at 2 x 106 cells in 60-mm dishes and treated with 0, 0.1, and 1 mmol/L VP-16 for 24 h. Total RNA was extracted by Trizol (Invitrogen) method according to the manufacturer's instructions. The RNA yield and purity were determined spectrophotometrically at 260 to 280 nm. The primers used for reverse transcription-PCR (RT-PCR) were as follows: 5'-GTCTTCGGGCCTGAGCTGTCG-3' and 5'-GGTTGTAGAATTAAGAATAGC-3' for TopII{alpha} and 5'-GGTCGGAGTCAACGGATTTG-3' and 5'-ATGAGCCCCAGCCTTCTCCAT-3' for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Drug accumulation, uptake, and efflux. Drug accumulation was determined by seeding 2 x 105cells onto 24-well plates. After 24 h, cells were washed with serum-free medium and incubated with 5 µmol/L [3H]VP-16 for 2 h. The reaction was stopped by adding ice-cold PBS and the cells were lysed immediately with 1% SDS for 30 min. The accumulation of the drug was measured by liquid scintillation counting.

Cell cycle analysis. Cells were plated and treated with the respective agents for the indicated times. Adherent and supernatant cells were collected, generated into a single-cell suspension, washed with PBS, and fixed with ice-cold 70% ethanol. Cells were then washed with PBS and treated with 5 µL of 1 mg/mL propidium iodide in 400 µL PBS for 30 min at room temperature. Propidium iodide incorporation was measured on a Becton Dickinson FACScan analyzer. Cells in sub-G0, G1, S, and G2-M were identified and quantified using quadrant statistics.

Immunoprecipitation. Cell lines were plated at 2 x 106 cells in T75 dishes. After the respective treatments, cells were harvested and lysed in lysis buffer containing 50 mmol/L Tris-HCl (pH 7.5), 1% sodium deoxycholate, 1% Triton X-100, 150 mmol/L NaCl, 10 µg/mL leupeptin, 10 µg/mL pepstatin, and 20 µg/mL aprotinin. The lysates were incubated with protein A plus G Sepharose beads and the respective antibody or isotype control overnight at 4°C. The beads were then washed thrice with PBS and boiled with 1x Laemmli buffer. The extracted proteins were loaded onto 6% SDS-PAGE gels followed by transfer to nitrocellulose membranes. The blots were assayed for the expression of TopoII{alpha} or MDM2 and enhanced by chemiluminescence detection (ECL).

Confocal imaging. Cells were plated at 2 x 106 cells in chamber slides and treated with 5 mmol/L VP-16 for 2 h. Cells were fixed for 20 min in 1:1 acetone/methanol at –20°C and treated with 5% bovine serum albumin to prevent nonspecific binding. Antibodies used were as follows: 1:100 anti-MDM2 (SMP14) and 1:100 anti-TopoII{alpha} (Ab-1). The samples were then washed thrice with PBS and incubated with Cy3-labeled antirabbit IgG and FITC-labeled antimouse IgG. After washing, samples were examined using a confocal laser scanning microscope (Zeiss LSM 510 META).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell lines homozygous for SNP309 have decreased sensitivity to TopoII-targeting drugs. It was reported previously that cell lines homozygous for MDM2 SNP309 had a decreased apoptotic response to DNA-damaging agents, such as VP-16 and gamma irradiation (20). We sought to determine whether this differential induction of apoptosis in cells homozygous for MDM2 SNP309 and those with WT MDM2 translated into differences in drug sensitivity to various classes of DNA-damaging agents. A panel of cell lines derived from various cell types and with varying status of p53 (Table 1 ) were treated with TopoII-targeting and topoisomerase I (TopoI)–targeting drugs and platinating agents. As shown in Fig. 1 , cells homozygous for SNP309 were ~10-fold resistant to TopoII-targeting drugs but were not resistant to other types of DNA-damaging agents (Fig. 1A–C); this effect was independent of the status of p53. SNP309 conferred resistance to multiple TopoII-targeting drugs, including VP-16, mitoxantrone, amsacrine, and ellipticine (Fig. 1D). In contrast, sensitivity to TopoI-targeting drugs and platinating agents was independent of MDM2 SNP309 but dependent on the status of p53; cell lines with WT p53 were more sensitive to DNA-damaging agents than those with mutant or null p53. Furthermore, MDM2 SNP309 had no effect on the sensitivity to non–DNA-damaging drugs, such as antimicrotubule agents in the select cell lines used (Supplementary Tables S1 and S2).


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  		Table 1. Status of MDM2 SNP309 and p53 status in cell lines

 

Figure 1
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  		Figure 1. Cells homozygous for SNP309 show decreased sensitivity to TopoII drugs. Cell lines WT (T/T; blue boxes) or homozygous for SNP309 (G/G; red triangles) were treated with increasing doses of VP-16 (A), camptothecin (B), cisplatin (C), and mitoxantrone, amsacrine, or ellipticine (D) for 48 h and cell viability was determined by MTT cell proliferation assay. Absorbances were determined at 570 nm using a Dynatech MR5000 plate reader. Viability was expressed as a percentage of control by dividing the absorbance of each treated well by the average of the untreated controls. IC50 was defined as the concentration of drug that decreased cell viability by 50%. Mean ± SD of triplicates (graph) and median (table) from four independent experiments.

 
TopoII{alpha} decreases in cells homozygous for SNP309 following treatment with VP-16. To explore the mechanism of resistance to TopoII-targeting drugs in cells homozygous for SNP309, we first addressed the possibility that differences were due to the expression of drug efflux proteins, such as P-gp, MRP1, or BCRP. As shown in Fig. 2A , there was no difference in expression of these proteins between cell lines homozygous for SNP309 and those with WT MDM2. There was also no difference in accumulation of VP-16 between cells homozygous for SNP309 and those with WT MDM2 (Fig. 2B). Hence, the difference in sensitivity to TopoII drugs was not attributable to changes in drug accumulation. We then compared the expression of TopoII{alpha} in cell lines homozygous for SNP309 and those with WT MDM2 following exposure to VP-16 using concentrations of VP-16 and timed exposure that caused <15% cell kill. Figure 3 shows that treatment with 1 µmol/L VP-16 for 24 h decreased TopoII{alpha} protein in cells homozygous for SNP309 but not in cells with WT MDM2 (Fig. 3A and B). This effect was independent of p53 status and not associated with decreased TopoII{alpha} mRNA (Fig. 3C).


Figure 2
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  		Figure 2. Resistance to TopoII-targeting drugs is not due to overexpression of drug efflux proteins. A, cell lines homozygous for SNP309 or WT MDM2 were assayed for the expression of the drug efflux proteins, P-gp, MRP-1, and BCRP, by Western blot as described in Materials and Methods. ß-Actin was used as a loading control. Data are representative of three independent experiments. B, the accumulation of [3H]VP-16 was determined as described in Materials and Methods. Columns, mean of quadruplicate determinations from one of three independent experiments; bars, SD.

 

Figure 3
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  		Figure 3. TopoII{alpha} is lowered following treatment with VP-16 in cells homozygous for SNP309 but not in cells with WT MDM2. Cell lines homozygous for SNP309 (A875, Manca, and T47D; A) and cell lines with WT MDM2 (H460, Tera 2, and Saos 2; B) were treated with VP-16 for 24 h and then assayed for the expression of TopoII{alpha} and MDM2 by Western blotting (WB). ß-Actin served as loading control. The blot is representative of three independent experiments. C, RT-PCR was done on Manca cells (SNP309) following exposure to VP-16 for 24 h. The RNA yield and purity were determined spectrophotometrically at 260 to 280 nm. The primers used for RT-PCR were as follows: 5'-GTCTTCGGGCCTGAGCTGTCG-3' and 5'-GGTTGTAGAATTAAGAATAGC-3' for TopoII{alpha} and 5'-GGTCGGAGTCAACGGATTTG-3' and 5'-ATGAGCCCCAGCCTTCTCCAT-3' for GAPDH. Data are representative of three independent experiments.

 
TopoII{alpha} expression varies during the cell cycle. It is expressed minimally during G1 and S and increases during transition to G2-M. Alterations in cell cycle due to overexpression of MDM2 therefore could account for reduced TopoII{alpha} in cells homozygous for SNP309. Therefore, we compared cell cycle distribution in cells with WT MDM2 to those homozygous for SNP309 before and after exposure to VP-16. We found that exposure to VP-16 in cells homozygous for SNP309 showed no evidence of apoptosis and resulted in accumulation of cells in S and G2-M, the two phases of the cell cycle where TopoII{alpha} expression is the highest (Supplementary Figs. S1 and S2). Thus, cell cycle alterations could not account for the lowered TopoII{alpha} expression or resistance to VP-16 in cells homozygous for SNP309.

Reciprocal relationship between TopoII{alpha} and MDM2 following treatment with VP-16. To examine the effect of VP-16 on TopoII{alpha}, MDM2, and p53, we treated A875 cells (homozygous for SNP309) with 1 µmol/L VP-16 for 24 h and used alkaline lysis to dissociate TopoII{alpha} from DNA. Figure 4A shows that TopoII{alpha} decreased by 1 h following treatment with VP-16. The decreased TopoII{alpha} coincided with increased expression of MDM2. p53 expression increased after 2 to 3 h of exposure, suggesting that p53 transcriptional repression was not likely to be responsible for the decreased TopoII{alpha}.


Figure 4
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  		Figure 4. Effect of VP-16 on TopoII{alpha}, MDM2, and p53. A, A875 cells were treated with 1 µmol/L VP-16 for 0, 1, 2, 3, 4, and 6 h and TopoII{alpha} and MDM2 protein expression was determined by Western blots. Lysates were prepared by alkaline lysis procedure as described previously (18). ß-Actin served as loading control. The blot is representative of three independent experiments. B, primary MEFs were plated at 2 x 106 cells in 60-mm dishes. Twenty-four hours after plating, cells were treated with 0, 0.1, 1, and 10 mmol/L VP-16 for 24 h. Cells were then harvested and lysed and 50 µg of total protein were loaded on a 6% SDS-PAGE gel. The gel was transferred to a nitrocellulose membrane and assayed for the expression of TopoII{alpha} and MDM2. ß-Actin served as loading control. C, MTT assay was done as described in Materials and Methods to determine the viability of MEFs following treatment with VP-16. Absorbances were determined at 570 nm using a Dynatech MR5000 plate reader. Viability was expressed as a percentage of control by dividing the absorbance of each treated well by the average of the untreated controls. IC50 was defined as the concentration of drug that decreased cell viability by 50%. Columns, mean of triplicates from four independent experiments; bars, SD.

 
To further explore this association between TopoII{alpha}, MDM2, and p53, primary MEFs WT (p53+/+; MDM2+/+), knockout for p53 (p53–/–; MDM2+/+), or knockout for p53 and MDM2 (p53–/–; MDM2–/–) were treated with increasing doses of VP-16 for 24 h. As shown in Fig. 4B, treatment with VP-16 reduced the cellular content of TopoII{alpha} in cells, in which both p53 and MDM2 were deleted (p53–/–; MDM2+/+). In contrast, VP-16 did not down-regulate TopII{alpha} in cells depleted of p53 alone (p53–/–; MDM2+/+) or in WT cells (p53+/+; MDM2+/+), suggesting that MDM2 was responsible, directly or indirectly, for the down-regulation of TopoII{alpha} following exposure to VP-16 (Fig. 4B). This observation was strengthened by the finding that knockout of both p53 and MDM2 resulted in IC50 values following exposure to VP-16 that were intermediate between those for WT and knockout for p53 alone cells (Fig. 4C).

Knockdown of MDM2 stabilizes TopoII{alpha} and decreases resistance to TopoII-targeting drugs. If MDM2 was responsible for the down-regulation of TopoII{alpha} following treatment with VP-16, we reasoned that depletion of MDM2 with siRNA would prevent the decrease of MDM2 and restore drug sensitivity. As shown in Fig. 5A , knockdown of MDM2 in untreated cells had minimal effect on TopoII{alpha}. Following treatment with VP-16, TopoII{alpha} decreased in control cells and in cells treated with nontargeting siRNA. In contrast, VP-16 failed to decrease TopoII{alpha} in cells depleted of MDM2 with siRNA. We next examined the effect of MDM2 siRNA on sensitivity to VP-16. As shown in Fig. 5B, silencing of MDM2 increased sensitivity to VP-16 by 2-fold. We observed this change in sensitivity in A875 and Manca cells, which are both homozygous for SNP309 (Fig. 5B). In contrast, there was no change in sensitivity to VP-16 in cell lines with WT MDM2 following treatment with MDM2 siRNA (Fig. 5C). These results suggest that the increased expression of MDM2 in cells homozygous for SNP309 is responsible for lowering TopoII{alpha} after treatment with VP-16.


Figure 5
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  		Figure 5. siRNA to MDM2 prevents loss of TopoII{alpha} following treatment with VP-16 and increases sensitivity to TopoII-targeting drugs. A, A875 cells were treated with 100 nmol/L siRNA to MDM2, Oligofectamine control, or 100 nmol/L nontargeting siRNA for 24 h. Cells were then treated with VP-16 for 24 h and assayed for expression of TopoII{alpha} and MDM2 by Western blot. The blot is representative of three independent experiments. B, A875 and Manca cells were treated with VP-16 for 48 h following depletion of MDM2 by siRNA or with the appropriate controls as described in Materials and Methods. MTT cell proliferation assay was done to determine viability following treatment with VP-16 in cells treated with Oligofectamine control, 100 nmol/L nontargeting (NT) siRNA, or 100 nmol/L siRNA to MDM2. Data are representative of four independent experiments. C, H460 cells were treated with VP-16 for 48 h following depletion of MDM2 by siRNA or with the appropriate controls as described in Materials and Methods. MTT cell proliferation assay was done to determine viability following treatment with VP-16 in cells treated with Oligofectamine control, 100 nmol/L nontargeting siRNA, or 100 nmol/L siRNA to MDM2. Data are representative of four independent experiments.

 
MDM2 interacts with TopoII{alpha} in vivo. MDM2 is known to interact with several proteins and regulate their activity. To determine whether MDM2 and TopoII{alpha} interact, we did coimmunoprecipitations using whole-cell lysates and antibodies specific for MDM2 or TopoII{alpha}. As shown in Fig. 6A , antibodies against MDM2 coimmunoprecipitated TopoII{alpha}. Furthermore, treatment of cells with VP-16 increased the association of TopoII{alpha} with MDM2. Reciprocal immunoprecipitation with TopoII{alpha} yielded similar results. Pretreatment with the proteasome inhibitor, MG132, enhanced the association of MDM2 with TopoII{alpha} (Fig. 6A).


Figure 6
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  		Figure 6. MDM2 associates with TopII{alpha} in vivo. A, coimmunoprecipitation of MDM2 and TopoII{alpha} was done in whole-cell extracts of A875 cells following exposure to 5 µmol/L VP-16 for 6 h or with prior sensitization to 5 µmol/L MG132 followed by 5 µmol/L VP-16 for 6 h. Immunoprecipitation (IP) was done with antibodies to MDM2 (SMP14), TopoII{alpha} (Ab-1), or mouse or rabbit IgG and followed by Western blotting with the reciprocal antibody. The blot is representative of three independent experiments. B and C, confocal microscopy was done using a Zeiss LSM 510 meta microscope before and 2 h after treatment of A875 cells with 5 µmol/L VP-16. The cells were then fixed and stained with FITC-labeled anti-MDM2 antibody and Cy3-labeled anti-TopoII{alpha} antibody. Results are representative of three identical experiments.

 
The interaction between TopoII{alpha} and MDM2 was further investigated by confocal imaging. In untreated cells, MDM2 showed a cytoplasmic and perinuclear distribution, whereas TopoII{alpha} was predominantly nuclear (Fig. 6B). Following exposure to VP-16, MDM2 increased in the cytoplasm where it colocalized with TopoII{alpha} (Fig. 6C), indicating shuttling of TopoII{alpha} by MDM2 from nucleus to the cytoplasm.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Since the sequencing of the human genome and the subsequent identification of ~4.5 million SNPs, there have been considerable efforts to link a SNP phenotype to sensitivity to certain chemotherapeutic drugs. The results presented in this report support the concept that a SNP in the MDM2 promoter results in resistance to a specific class of chemotherapeutic agent (i.e., the TopoII-targeting drugs). The increased expression of MDM2 in cell lines homozygous for SNP309 results in lowered TopoII, the target of TopoII drugs, following drug exposure. Decreased expression of TopoII{alpha} was shown previously to be a mechanism of resistance to TopoII-targeting drugs (21, 22). Our data extend those of Bond et al. (20) and provide a mechanistic basis for the finding that MDM2 SNP309 results in attenuation of the response to VP-16.

Decreased sensitivity to TopoII-targeting drugs might be attributed to one of several mechanisms. For example, increased expressions of drug efflux proteins, such as P-gp, MRP1, or BCRP, could result in decreased drug accumulation and increased resistance to certain TopoII-targeting agents. Overexpression of MDM2 has been reported to result in increased expression of the MDR1 gene and P-gp in human glioblastoma cells, thus resulting in resistance to VP-16 and doxorubicin (15). However, the role of MDM2 in sensitivity to TopoII-targeting agents is controversial. For example McKenzie et al. (16) showed that MDM2 does not influence sensitivity to DNA-damaging agents. We found that overexpression of MDM2 in cell lines homozygous for SNP309 does not result in overexpression of drug efflux proteins, such as P-gp, MRP1, or BCRP (Fig. 2A). There is also no significant difference in accumulation of [3H]VP-16 between cell lines with WT MDM2 and those homozygous for SNP309 (Fig. 2B). Thus, the involvement of drug efflux in difference in sensitivities to TopoII-targeting agents in this context can be excluded. Although resistance to VP-16 mediated by MDM2 SNP309 is moderate (10-fold) in comparison with other acquired mechanisms of drug resistance, this SNP is a germ-line mutation and therefore would also affect the sensitivity of normal cells to certain drugs. The 10-fold increase in drug resistance produced by SNP309 would have major implications in the clinic, where cytotoxic drugs, like topoisomerase-targeting agents, are administered at or near the maximum tolerated dose.

TopoII-targeting agents exert their effect by "poisoning" TopoII{alpha}, a nuclear enzyme that modifies the tertiary structure of DNA. Hence, cells with lower TopoII{alpha} or with altered function of TopoII{alpha} due to mutations are resistant to TopoII-targeting drugs (21–23). We show that following exposure to concentrations of VP-16 that cause less than 15% cell killing, cell lines homozygous for SNP309 down-regulate TopoII{alpha} more efficiently than cell lines with WT MDM2 (Fig. 3A and B). This pointed to a previously unappreciated relationship between MDM2 and TopoII{alpha}. It has been shown that p53 can transcriptionally repress TopoII{alpha} (24). However, we found that the resistance to TopoII-targeting agents in cell lines homozygous for SNP309 occurs independent of p53 status (Fig. 3A and B). Moreover, the lowered TopoII{alpha} protein is seen without changes in its mRNA (Fig. 3C).

TopoII{alpha} protein expression varies during the cell cycle (25). Therefore, we also ruled out the possibility that the decreased sensitivity to TopoII-targeting drugs was due to decreased TopoII{alpha} expression in SNP309 cells due to cell cycle changes. In most cells, the expression of TopoII{alpha} becomes detectable by late G1 phase and peaks in G2-M. MDM2 is known to play an important role in progression through the cell cycle (10). It controls the fate of several key proteins in the cell cycle, such as p53 (8) and Rb (12), by promoting proteasomal-mediated degradation of these two proteins. However, MDM2 is also known to enhance the function of S phase-promoting factor E2F1 (13). It was conceivable that following DNA damage with VP-16, overexpression of MDM2 in cell lines homozygous for SNP309 could result in altered cell cycle distribution that would favor a lower cellular content of TopoII{alpha}. In contrast, we observed that in cell lines homozygous for SNP309, following exposure to 1 µmol/L VP-16, most cells accumulate in G2-M phase where TopoII{alpha} expression is maximum (Supplementary Figs. S1 and S2).

Having ruled out several explanations for our observations, we focused on the possible regulation of TopoII{alpha} by MDM2. We found that VP-16–mediated repression of TopoII{alpha} is dependent on the increased expression of MDM2. Thus, when MDM2 is depleted by siRNA, the levels of TopoII{alpha} do not change following exposure to VP-16 and sensitivity to the drug is increased compared with SNP209 cells treated with nontargeting siRNA controls (Fig. 5). The lack of total reversal of VP-16 resistance is likely due to the inability to completely restore TopoII{alpha} activity by knockdown of MDM2, or MDM2 silencing-induced increases in expression of p53, which is known to transcriptionally repress TopoII{alpha} expression (26).

It is known that MDM2 shuttles between the nucleus and the cytoplasm (27, 28). For example, Freedman and Levine (27) showed that the regulation of p53 level by MDM2 requires the nuclear export activity of MDM2. We show that a physical interaction occurs between MDM2 and TopoII{alpha}. We find that MDM2 and TopoII{alpha} coimmunoprecipitate and colocalize in cancer cells (Fig. 6). The cellular distribution of TopoII{alpha} and MDM2 changes following exposure to VP-16 when the two proteins are located within the cytoplasm. These results suggest that export of TopoII{alpha} by MDM2 from the nucleus to the cytoplasm might be responsible for the down-regulation of TopoII{alpha}. Furthermore, the association is enhanced following treatment with VP-16 (Fig. 6A). MG132, a proteasome inhibitor, also enhances the association between the two proteins, suggesting that the increased MDM2 due to inhibition of the ubiquitin pathway might lead to increased association between MDM2 and TopoII{alpha} (Fig. 6A).

The transcription factor Sp1 is known to activate both TopoII{alpha} and MDM2. It has been shown previously that increased expression of MDM2 in cell lines homozygous for SNP309 is due to increased binding of Sp1 to the MDM2 promoter. Johnson-Pais et al. (29) have shown that MDM2 binding to Sp1 prevents its specific DNA binding to other sequences. Further, MDM2 and TopoII{alpha} commonly interact with several proteins, such as Rb.

The precise mechanism by which the association of MDM2 with TopoII{alpha} leads to down-regulation of the drug target remains unclear. It is likely that post-translational events following DNA damage with VP-16 are important. For example, following exposure to VP-16, several kinases, such as ATM, phosphorylate MDM2 and TopoII{alpha}. Post-translational modification, such as phosphorylation of the androgen receptor, enhances degradation by MDM2 (30). In addition, ATM-mediated phosphorylation of Mdmx by ATM mediates its degradation by MDM2 following DNA damage (31). There are also instances of MDM2-mediated degradation of proteins independent of ubiquitination. MDM2 interacts with p21 and facilitates its interaction with C8 subunit of 20S proteasome independent of p53 and ubiquitination (13).

In this study, we confirm and extend the observations of Bond et al. (20) and strengthen the correlation between a SNP phenotype and sensitivity to chemotherapeutic agents. Given the frequency of SNP309 in the general population, these data may have important implications for the individualization of cancer chemotherapy.


   Acknowledgments
 
Grant support: U.S. Health Service grants CA 78695 and CA 72720.

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.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 12/ 8/06. Revised 3/12/07. Accepted 4/23/07.


   References
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 Abstract
 Introduction
 Materials and Methods
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 References
 

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