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Antisense imaging of epidermal growth factor-induced p21WAF-1/CIP-1 gene expression in MDA-MB-468 human breast cancer xenografts

  • Molecular Imaging
  • Published:
European Journal of Nuclear Medicine and Molecular Imaging Aims and scope Submit manuscript

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 EGF-induced p21WAF-1/CIP-1 gene expression in MDA-MB-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.

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Acknowledgements

This study was supported by grants from the Cancer Research Society Inc., the Breast Cancer Society of Canada, and the Ontario Research and Development Challenge Fund. Meiduo Hu is the recipient of a predoctoral training award from the U.S. Army Breast Cancer Research Program (DAMD17-02-1-0598). Parts of this study were presented at the Society of Nuclear Medicine 49th Annual Meeting, Los Angeles, CA, June 15–19, 2002. The authors acknowledge the expert technical assistance of Robert Kamen and Deborah Scollard in the acquisition of the images and Shaoxian Yang and Quinghong Zhang for valuable advice in RT-PCR analyses.

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Authors and Affiliations

  1. Leslie Dan Faculty of Pharmacy, University of Toronto, 19 Russell Street, Toronto, Ontario, M5S 2S2, Canada

    Judy Wang, Paul Chen, Marko Mrkobrada, Meiduo Hu & Raymond M. Reilly

  2. Department of Radiation Oncology, Princess Margaret Hospital, University Health Network, 610 University Avenue, Toronto, Ontario, Canada

    Katherine A. Vallis

  3. Department of Radiation Oncology, University of Toronto, Toronto, Ontario, Canada

    Katherine A. Vallis

  4. Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada

    Katherine A. Vallis

  5. Department of Medical Imaging, University of Toronto, Toronto, Ontario, Canada

    Raymond M. Reilly

  6. Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada

    Meiduo Hu & Raymond M. Reilly

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  1. Judy Wang
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  2. Paul Chen
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Correspondence to Raymond M. Reilly.

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Wang, J., Chen, P., Mrkobrada, M. et al. Antisense imaging of epidermal growth factor-induced p21WAF-1/CIP-1 gene expression in MDA-MB-468 human breast cancer xenografts. Eur J Nucl Med Mol Imaging 30, 1273–1280 (2003). https://doi.org/10.1007/s00259-003-1134-0

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  • DOI: https://doi.org/10.1007/s00259-003-1134-0

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