Molecular imaging Antisense imaging of epidermal growth factor-induced p21WAF-1/CIP-1 gene expression in MDA-MB-468 human breast cancer xenografts Judy Wang1, Paul Chen1, Marko Mrkobrada1, Meiduo Hu1, 6, Katherine A. Vallis2, 3, 4, Raymond M. Reilly5, 6 1 Leslie
Dan Faculty of Pharmacy, University of Toronto, 19 Russell Street, Toronto, Ontario, M5S 2S2 Canada of Radiation Oncology, Princess Margaret Hospital, University Health Network, 610 University Avenue, Toronto, Ontario, Canada 3 Department of Radiation Oncology, University of Toronto, Toronto, Ontario, Canada 4 Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada 5 Department of Medical Imaging, University of Toronto, Toronto, Ontario, Canada 6 Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada 2 Department
Published online: 15 March 2003 © Springer-Verlag 2003
Abstract. Molecular imaging of the expression of key genes which determine the response to DNA damage following cancer treatment may predict the effectiveness of a particular treatment strategy. A prominent early response gene for DNA damage is the gene encoding p21WAF-1/CIP-1, a cyclin-dependent kinase inhibitor that regulates progression through the cell cycle. In this study, we explored the feasibility of imaging p21WAF-1/CIP-1 gene expression at the mRNA level using an 18-mer phosphorothioated antisense oligodeoxynucleotide (ODN) labeled with 111In. The known induction of the p21WAF-1/CIP-1 gene in MDA-MB-468 human breast cancer cells following exposure to epidermal growth factor (EGF) was used as an experimental tool. Treatment of MDA-MB-468 cells in vitro with EGF (20 nM) increased the ratio of p21WAF-1/CIP-1 mRNA/β-actin mRNA threefold within 2 h as measured by the reverse transcription polymerase chain reaction (RT-PCR). A concentration-dependent inhibition of EGF-induced p21WAF-1/CIP-1 protein expression was achieved in MDA-MB-468 cells by treatment with antisense ODNs with up to a tenfold decrease observed at 1 µM. There was a fourfold lower inhibition of p21WAF-1/CIP-1 protein expression by control sense or random sequence ODNs. Intratumoral injections of EGF (15 µg/day×3 days) were employed to induce p21WAF-1/CIP-1 gene expression in MDA-MB-468 xenografts implanted subcutaneously into athymic mice. RT-PCR of explanted tumors showed a threefold increased level of p21WAF-1/CIP-1 mRNA compared with normal saline-treated tumors. Successful imaging of Raymond M. Reilly (✉) Leslie Dan Faculty of Pharmacy, University of Toronto, 19 Russell Street, Toronto, Ontario, M5S 2S2, Canada e-mail:
[email protected] Tel.: +1-416-9465522, Fax: +1-416-9788511
EGF-induced p21WAF-1/CIP-1 gene expression in MDAMB-468 xenografts was achieved at 48 h post injection of 111In-labeled antisense ODNs (3.7 MBq; 2 µg). Tumors displaying basal levels of p21WAF-1/CIP-1 gene expression in the absence of EGF treatment could not be visualized. Biodistribution studies showed a significantly higher tumor accumulation of 111In-labeled antisense ODNs in the presence of EGF induction of the p21WAF-1/CIP-1 gene (0.32%±0.06% injected dose/g) compared with normal saline-treated control mice (0.11%±0.07% injected dose/g). The tumor/blood ratio for antisense ODNs in the presence of EGF induction of the p21WAF-1/CIP-1 gene (4.87±0.87) was also significantly higher than for control random sequence ODNs (2.14±0.69) or for mice receiving antisense ODNs but not treated with EGF (2.07±0.37). We conclude that antisense imaging of upregulated p21WAF-1/CIP-1 gene expression is feasible and could represent a promising new molecular imaging strategy for monitoring tumor response in cancer patients. To our knowledge, this study also describes the first report of molecular imaging of the upregulated expression of a downstream gene target of the EGFR, a transmembrane tyrosine kinase receptor. Keywords: Antisense oligonucleotides – Indium-111 – Epidermal growth factor – p21WAF-1/CIP-1 – Breast cancer Eur J Nucl Med (2003) 30:1273–1280 DOI 10.1007/s00259-003-1134-0
Introduction Molecular imaging of the expression of key genes regulating the response to DNA damage may allow non-inva-
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sive probing of early molecular events following cancer treatment. If successful, it could possibly predict the effectiveness of a particular treatment strategy or resistance to the treatment in an individual patient. Chemotherapeutic agents and gamma radiation are thought to produce cytotoxic effects on cancer cells by causing DNA damage activating the apoptotic cascade [1, 2]. A key mediator of the early molecular response to DNA damage is p53, a nuclear transcription factor that regulates the expression of many downstream genes, including the cyclin-dependent kinase (cdk) inhibitor, p21WAF-1/CIP-1 [3]. p21WAF-1/CIP-1 specifically binds and inactivates cyclin-D/cdk-4 and cyclin-E/cdk-2 complexes, causing cell cycle arrest at the G1/S phase checkpoint or promotion of apoptosis in cells with irreparably damaged DNA [3, 4]. Mutations in the p53 gene are the most common genetic abnormality in cancer [5, 6] and have been implicated in resistance to treatment by preventing a normal p21WAF-1/CIP-1 response [7]. p21WAF-1/CIP-1 gene expression is also upregulated by a p53-independent mechanism in MDA-MB-468 breast cancer cells or A431 epidermoid carcinoma cells [8, 9]. Despite the expression of high levels of epidermal growth factor receptors (EGFR), these cells are growth-inhibited by supraphysiological concentrations of EGF. The antiproliferative effects are mediated by EGF-induced upregulation of the p21WAF-1/CIP-1 gene, producing cell cycle arrest and apoptosis [8, 9]. In this study, we exploited the known EGF-induced expression of the p21WAF-1/CIP-1 gene in MDA-MB-468 human breast cancer cells as an experimental tool to study the potential for imaging upregulated p21WAF-1/CIP-1 gene expression using a radiolabeled DNA antisense oligonucleotide. Materials and methods Breast cancer cells. MDA-MB-468 human breast cancer cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were cultured in DMEM (Ontario Cancer Institute, Toronto, ON) containing 100 units/ml of penicillin, 100 µg/ml of streptomycin, and 2 mM L-glutamine and supplemented with 10% fetal calf serum (Sigma Chemical Co., St. Louis, MO). MDA-MB-468 cells express 1–2×106 EGFR/cell [10]. DNA oligonucleotides. A fully phosphorothioated 18-mer antisense DNA oligonucleotide (ODN) consisting of the sequence 5’-AGCCGGTTCTGACATGGC-3’, previously described by Ohtsubo et al. [11] and corresponding to nucleotides 51–68 at the 5’-end of the p21WAF-1/CIP-1 gene [12], and the corresponding sense sequence ODN, 5’-GCCATGTCAGAACCGGCT-3’, were synthesized using phosphoramidite chemistry (Gibco Life Technologies, Burlington, ON). An 18-mer ODN with the random sequence 5’-ACGCGTTGACTGCCTAGG-3’, containing the same proportion of constituent nucleotides, was also constructed. ODNs were synthesized with an amino group linked to the 5’ sugar through a six carbon spacer (aminohexyl linker) for derivatization with diethylene triamine penta-acetic acid (DTPA) and subsequent label-
ing with 111In. The random sequence was not homologous with any sequence in the human genome evaluated using the Nucleotide BLAST™ database [13]. Radiolabeling of oligonucleotides. The aminohexyl-containing ODNs were dissolved at 5 mg/ml in 50 mM sodium bicarbonate in 150 mM sodium chloride buffer pH 7.5 and reacted with a 100fold molar excess of the bicyclic anhydride of DTPA (Sigma) at room temperature for 30 min as described by Dewanjee et al. [14]. DTPA-derivatized ODNs were purified by size-exclusion chromatography on a P-2 mini-column (BioRad, Mississauga, ON) eluted with 150 mM sodium chloride. Purified DTPA-derivatized oligonucleotides were labeled with 111In to a specific activity of approximately 1.4 MBq/µg (8,100 MBq/µmol) by incubation with 111In acetate for 30 min at room temperature. 111In acetate was prepared by mixing 111In chloride (MDS-Nordion, Kanata, ON) with 1 M sodium acetate buffer pH 6. The radiochemical purity of 111In-labeled ODNs was 95–98% as determined by silica gel instant thinlayer chromatography (ITLC-SG, Gelman, Ann Arbor, MI) developed in 100 mM sodium citrate pH 5.0. Kinetics of EGF-induced p21WAF-1/CIP-1 gene expression. The kinetics of EGF-induced p21WAF-1/CIP-1 gene expression in MDAMB-468 cells were evaluated by the reverse transcription polymerase chain reaction (RT-PCR). Approximately 1×106 MDA-MB-468 cells were seeded into 60-mm culture dishes (Nunc, Gibco Life Technologies) and treated for 30 min, 1 h, 2 h, or 4 h with 20 nM of human EGF (Upstate Biotechnology, Lake Placid, NY) in culture medium at 37°C. Total RNA was extracted using the RNeasy MiniKit (Quiagen). First strand cDNA was directly prepared from 1 µg of RNA by reverse transcription using random hexadeoxynucleotide primers (Gibco Life Technologies) and 200 units/ml of Superscript II reverse transcriptase (Gibco Life Technologies). Primers for p21WAF-1/CIP-1 or β-actin genes [15] were utilized to amplify the respective cDNAs by PCR. The PCR conditions were: 94°C for 20 s, 55°C for 20 s and 72°C for 45 s. A total of 27 cycles were performed for β-actin and 32 cycles for p21WAF-1/CIP-1. The PCR products were analyzed on a 2% agarose gel containing 100 µg/ml of ethidium bromide and bands quantified by gel densitometry using image analysis software (LabWorks TM Analysis, DiaMed, Mississauga, ON). Antisense inhibition of EGF-induced p21WAF-1/CIP-1 gene expression. The functional activity of the aminohexyl linked antisense ODNs was evaluated by studying their ability to inhibit p21WAF-1/CIP-1 protein translation in MDA-MB-468 cells determined by Western blot. Approximately 5×105 MDA-MB-468 cells were cultured overnight in 60-mm dishes. The cells were treated for 5 h at 37°C with 50, 100, or 500 nM or 1 µM of aminohexyl linked p21WAF-1/CIP-1 antisense ODNs or 1 µM of the corresponding sense or random sequence ODNs in OptiMem containing 20 µg/ml of Lipofectin. The cells were cultured overnight with 20 nM of EGF at 37°C to induce p21WAF-1/CIP-1 gene expression. The cells were then lysed in a buffer consisting of 0.1% sodium dodecylsulfonate, 0.1% sodium deoxycholate, 0.1% Triton X-100 in 10 mM Tris HCl in 150 mM sodium chloride pH 7.4 containing proteinase inhibitor (Roche, Montreal, PQ). Sodium dodecylsulfonate polyacrylamide gel electrophoresis (SDS-PAGE) was performed under reducing conditions on 10 µl of cell lysates on a 4–20% Tris HCl gradient Ready-Gel (BioRad, Mississauga, ON). Electrophoresed proteins were transferred to a nitrocellulose membrane (Trans-Blot, BioRad) using 25 mM Tris, 192 mM glycine pH 8.3 buffer. The membranes were blocked overnight with 5%
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1275 Carnation skimmed milk in Tris-buffered saline (TBS: 150 mM sodium chloride, 20 mM Tris-HCl and 0.1% Tween-20, pH 7.5), rinsed with TBS, and then incubated at 37°C for 45 min with 1 µg/ml mouse anti-p21WAF-1/CIP-1 antibodies (Oncogene Research, Cedarlane Laboratories, Misssissauga, ON) or 1 µg/ml β-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were rinsed with TBS and then incubated with a 1:1,500 dilution of goat anti-mouse antibody-HRP conjugate (Sigma) in TBS at 37°C for 45 min. The membranes were rinsed again and reactive bands detected using the enhanced chemiluminescence kit (ECL kit, NEN Life Science Products, Boston, MA). Bands were visualized by exposure of the membranes to Kodak BioMax Imaging film (Kodak, Rochester, NY) and the intensity of p21WAF-1/CIP-1 and β-actin bands quantified by gel densitometry. Antisense imaging of p21WAF-1/CIP-1 gene expression. The potential for imaging EGF-induced p21WAF-1/CIP-1 gene expression in vivo using 111In-labeled antisense ODNs was investigated in athymic mice bearing s.c. MDA-MB-468 xenografts. Tumors were established in female Swiss athymic (nu/nu) mice by s.c. injection of 1×107 MDA-MB-468 cells in the left hind leg. After 4–6 weeks, when tumors were approximately 0.25 cm in diameter, the mice were injected intratumorally with 15 µg EGF in 50 µl of normal saline for 3 consecutive days to upregulate tumor p21WAF-1/CIP-1 gene expression or an equivalent amount of normal saline (control). The mice were then injected i.v. (tail vein) with 0.37–3.7 MBq (0.2–2 µg) of 111In-labeled p21WAF-1/CIP-1 antisense, sense, or random sequence ODNs. Posterior whole-body images were obtained at 48 h post injection of 111In-labeled p21WAF-1/CIP-1 antisense ODNs (with/without EGF-induced gene expression) on a Siemens ZLC-3700 gamma camera fitted with a pinhole collimator. The mice were then sacrificed by cervical dislocation and samples of tumor and normal tissues, including blood, were obtained, weighed, and counted along with a standard of the injected radiopharmaceuticals in a gamma counter. Tumor and normal tissue uptake of 111In was expressed as percent injected dose/g (% i.d./g) and as tumor/normal tissue (T/NT) ratios. Induction of the p21WAF-1/CIP-1 gene in vivo by EGF was evaluated by analysis of tumor mRNA by RT-PCR as previously described for MDAMB-468 cells. The Principles of Laboratory Animal Care (NIH Publication No. 86-23, revised 1985) were followed and animal studies were conducted under a protocol approved by the Animal Care Committee at the University Health Network (No. TG-01052) and following Canadian Council on Animal Care (CCAC) guidelines. Statistical comparisons. Data were expressed as mean±SEM. Statistical comparisons were made by ANOVA and post-testing was performed using Dunn’s Multiple Comparisons Test (P0.05), the tumor/blood (T/B) ratio for the antisense ODNs (Table 2) was significantly greater (4.87±0.87 vs 2.14±0.69; P
