title: Antisense imaging of epidermal growth factor-induced p21(WAF-1/CIP-1) gene expression in MDA-MB-468 human breast cancer xenografts.
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 p21(WAF-1/CIP-1), a cyclin-dependent kinase inhibitor that regulates progression through the cell cycle. In this study, we explored the feasibility of imaging p21(WAF-1/CIP-1) gene expression at the mRNA level using an 18-mer phosphorothioated antisense oligodeoxynucleotide (ODN) labeled with (111)In. The known induction of the p21(WAF-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 n M) increased the ratio of p21(WAF-1/CIP-1) mRNA/beta-actin mRNA threefold within 2 h as measured by the reverse transcription polymerase chain reaction (RT-PCR). A concentration-dependent inhibition of EGF-induced p21(WAF-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 microM. There was a fourfold lower inhibition of p21(WAF-1/CIP-1) protein expression by control sense or random sequence ODNs. Intratumoral injections of EGF (15 microg/dayx3 days) were employed to induce p21(WAF-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 p21(WAF-1/CIP-1) mRNA compared with normal saline-treated tumors. Successful imaging of EGF-induced p21(WAF-1/CIP-1) gene expression in MDA-MB-468 xenografts was achieved at 48 h post injection of (111)In-labeled antisense ODNs (3.7 MBq; 2 microg). Tumors displaying basal levels of p21(WAF-1/CIP-1) gene expression in the absence of EGF treatment could not be visualized. Biodistribution studies showed a significantly higher tumor accumulation of (111)In-labeled antisense ODNs in the presence of EGF induction of the p21(WAF-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 p21(WAF-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 p21(WAF-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.
authors.affiliation: Leslie Dan Faculty of Pharmacy, University of Toronto, M5S 2S2 Ontario, Toronto, Canada.
authors.forename: Judy
authors.initials: J
authors.lastname: Wang
authors.forename: Paul
authors.initials: P
authors.lastname: Chen
authors.forename: Marko
authors.initials: M
authors.lastname: Mrkobrada
authors.forename: Meiduo
authors.initials: M
authors.lastname: Hu
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Vallis
authors.forename: Raymond M
authors.initials: RM
authors.lastname: Reilly
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000000981:diagnostic imaging* / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000378:metabolism* / Q000494:pharmacology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007205:Indium Radioisotopes / Q000493:pharmacokinetics; D007553:Isotope Labeling / Q000379:methods; D008657:Metabolic Clearance Rate; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000493:pharmacokinetics*; D009928:Organ Specificity; D011877:Radionuclide Imaging; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics; D014018:Tissue Distribution; D014183:Transplantation, Heterologous; D014915:Whole-Body Counting
chemical_list: C493956:CDKN1A protein, human; C493957:Cdkn1a protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D007205:Indium Radioisotopes; D016376:Oligonucleotides, Antisense; D019275:Radiopharmaceuticals; D004815:Epidermal Growth Factor