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123I-labeled HIV-1 tat peptide radioimmunoconjugates are imported into the nucleus of human breast cancer cells and functionally interact in vitro and in vivo with the cyclin-dependent kinase inhibitor, p21WAF-1/Cip-1

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

Abstract

Purpose

To evaluate the internalization and nuclear translocation of 123I-tat-peptide radioimmunoconjugates in MDA-MB-468 breast cancer cells and their ability to interact with the cyclin-dependent kinase inhibitor, p21WAF-1/Cip-1.

Methods

Peptides [GRKKRRQRRRPPQGYGC] harboring the nuclear-localizing sequence from HIV tat domain were conjugated to anti-p21WAF-1/Cip-1 antibodies. Immunoreactivity was assessed by Western blot using lysate from MDA-MB-468 cells exposed to EGF to induce p21WAF-1/Cip-1. Internalization and nuclear translocation were measured. The ability of tat-anti-p21WAF-1/Cip-1 to block G1-S phase arrest in MDA-MB-468 cells caused by EGF-induced p21WAF-1/Cip-1 was evaluated. Tumor and normal tissue uptake were determined at 48 h p.i. in athymic mice implanted s.c. with MDA-MB-468 xenografts injected intratumorally with EGF.

Results

There was 13.4±0.2% of radioactivity internalized by MDA-MB-468 cells incubated with 123I-tat-anti-p21WAF-1/Cip-1 and 34.6±3.1% imported into the nucleus. Tat-anti-p21WAF-1/Cip-1(8 μM) decreased the proportion of EGF-treated cells in G1 phase from 81.9±0.7% to 46.1±0.7% (p<0.001), almost restoring the G1 phase fraction to that of unexposed cells (25.8±0.2%). Non-specific tat-mouse IgG did not block EGF-induced G1-S phase arrest. Tumor uptake of radioactivity was higher in mice injected with EGF to induce p21WAF-1/Cip-1 than in mice not receiving EGF (3.1±0.4% versus 1.8±0.2% ID/g; p=0.04). Western blot analysis of tumors revealed a threefold increase in the p21WAF-1/Cip-1/β-actin ratio.

Conclusion

We conclude that intracellular and nuclear epitopes in cancer cells can be functionally targeted with tat-radioimmunoconjugates to exploit many more epitopes for imaging and radiotherapeutic applications than have previously been accessible.

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References

  1. DeNardo SJ. Radioimmunodetection and therapy of breast cancer. Semin Nucl Med 2005;35:143–51.

    Article  PubMed  Google Scholar 

  2. Sharkey RM, Goldenberg DM. Perspectives on cancer therapy with radiolabeled monoclonal antibodies. J Nucl Med 2005;46:115S–127S.

    PubMed  CAS  Google Scholar 

  3. Reilly RM. Biomolecules as targeting vehicles for in situ radiotherapy of malignancies. In: Knaeblein J, ed. Modern biopharmaceuticals: design, development and optimization., vol. 2. Weinheim, Germany: Wiley-VCH; 2005. pp. 497–526.

    Google Scholar 

  4. Misek DE, Imafuku Y, Hanash SM. Application of proteomic technologies to tumor analysis. Pharmacogenomics 2004;5:1129–37.

    Article  PubMed  CAS  Google Scholar 

  5. Bargou RC, Daniel PT, Mapara MY, Bommert K, Wagener C, Kallinich B, et al. Expression of the Bcl-2 gene family in normal and malignant breast tissue: low bax-α expression in tumor cells correlates with resistance towards apoptosis. Int J Cancer 1995;60:854–9.

    PubMed  CAS  Google Scholar 

  6. Brodeur GM, Seeger RC, Schwab M, Varmus HE, Bishop JM. Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage. Science 1984;224:1121–4.

    Article  PubMed  CAS  Google Scholar 

  7. Sigal A, Rotter V. Oncogenic mutations of the p53 tumor suppressor: the demons of the guardian of the genome. Cancer Res 2000;60:6788–93.

    PubMed  CAS  Google Scholar 

  8. Hu M, Chen P, Wang J, Chan C, Scollard DA, Reilly RM. Site-specific conjugation of HIV-1 tat peptides to IgG: a potential route to construct radioimmunoconjugates for targeting intracellular and nuclear epitopes in cancer. Eur J Nucl Med Mol Imag 2005;33:301–10.

    Article  CAS  Google Scholar 

  9. Rodriguez-Vilarrupla A, Diaz C, Canela N, Rahn HP, Bachs O, Agell N. Identification of the nuclear localization signal of p21cip1 and consequences of its mutation on cell proliferation. FEBS Lett 2002;531:319–23.

    Article  PubMed  CAS  Google Scholar 

  10. Taules M, Rodriguez-Vilarrupla A, Rius E, Estanyol JM, Casanovas O, Sacks DB, et al. Calmodulin binds to p21Cip1 and is involved in the regulation of its nuclear localization. J Biol Chem 1999;274:24445–8.

    Article  PubMed  CAS  Google Scholar 

  11. el-Deiry WS, Harper JW, O’Connor PM, Velculescu VE, Canman CE, Jackman J, et al. WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res 1994;54:1169–74.

    PubMed  CAS  Google Scholar 

  12. Gorospe M, Wang X, Holbrook NJ. Functional role of p21 during the cellular response to stress. Gene Expr 1999;7:377–85.

    PubMed  CAS  Google Scholar 

  13. Fang M, Liu B, Schmidt M, Lu Y, Mendelsohn J, Fan Z. Involvement of p21Waf1 in mediating inhibition of paclitaxel-induced apoptosis by epidermal growth factor in MDA-MB-468 human breast cancer cells. Anticancer Res 2000;20:103–11.

    PubMed  CAS  Google Scholar 

  14. Liu W, Zhang R. Upregulation of p21WAF1/CIP1 in human breast cancer cell lines MCF-7 and MDA-MB-468 undergoing apoptosis induced by natural product anticancer drugs 10-hydroxycamptothecin and camptothecin through p53-dependent and independent pathways. Int J Oncol 1998;12:793–804.

    PubMed  CAS  Google Scholar 

  15. Fan Z, Lu Y, Wu XP, Deblasio A, Koff A, Mendelsohn J. Prolonged induction of p21Cip1/WAF1/CDK2/PCNA complex by epidermal growth factor receptor activation mediates ligand-induced A431 cell growth inhibition. J Cell Biol 1995;131:235–42.

    Article  PubMed  CAS  Google Scholar 

  16. Armstrong DK, Kaufmann SH, Ottaviano YL, Furuya Y, Buckley JA, Isaacs JT, et al. Epidermal growth factor-mediated apoptosis of MDA-MB-468 human breast cancer cells. Cancer Res 1994;54:5280–3.

    PubMed  CAS  Google Scholar 

  17. Thomas T, Balabhadrapathruni S, Gardner CR, Hong J, Faaland CA, Thomas TJ. Effects of epidermal growth factor on MDA-MB-468 breast cancer cells: alterations in polyamine biosynthesis and the expression of p21/CIP1/WAF1. J Cell Physiol 1999;179:257–66.

    Article  PubMed  CAS  Google Scholar 

  18. Fawell S, Seery J, Daikh Y, Moore C, Chen LL, Pepinsky B, et al. Tat-mediated delivery of heterologous proteins into cells. Proc Natl Acad Sci USA 1994;91:648–64.

    Article  Google Scholar 

  19. Wang J, Chen P, Mrkobrada M, Hu M, Vallis KA, Reilly RM. 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 2003;30:1273–80.

    Article  PubMed  Google Scholar 

  20. Vocero-Akbani AM, Heyden NV, Lissy NA, Ratner L, Dowdy SF. Killing HIV-infected cells by transduction with an HIV protease-activated caspase-3 protein. Nat Med 1999;5:29–33.

    Article  PubMed  CAS  Google Scholar 

  21. Nagahara H, Vocero-Akbani AM, Snyder EL, Ho A, Latham DG, Lissy NA, et al. Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nat Med 1998;4:1449–52.

    Article  PubMed  CAS  Google Scholar 

  22. Schwarze SR, Ho A, Vocero-Akbani AM, Dowdy SF. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 1999;285:1569–72.

    Article  PubMed  CAS  Google Scholar 

  23. Caron NJ, Torrente Y, Camirand G, Bujold M, Chapdelaine P, Leriche K, et al. Intracellular delivery of a Tat-eGFP fusion protein into muscle cells. Mol Ther 2001;3:310–8.

    Article  PubMed  CAS  Google Scholar 

  24. Zhao M, Kirscher MF, Josephson L, Weissleder R. Differential conjugation of tat peptide to supermagnetic nanoparticles and its effect on cellular uptake. Bioconjug Chem 2002;13:840–4.

    Article  PubMed  CAS  Google Scholar 

  25. Zhang YM, Liu CB, Liu N, Ferro Flores G, He J, Rusckowski M, et al. Electrostatic binding with tat and other cationic peptides increases cell accumulation of 99mTc-antisense DNAs without entrapment. Mol Imaging Biol 2003;5:240–7.

    Article  PubMed  Google Scholar 

  26. Niesner U, Halin C, Lozzi L, Günthert M, Neri P, Wunderli-Allenspach H, et al. Quantitation of the tumor-targeting properties of antibody fragments conjugated to cell-permeating HIV-1 TAT peptides. Bioconjug Chem 2002;13:729–36.

    Article  PubMed  CAS  Google Scholar 

  27. Reilly R. The immunoreactivity of radiolabeled antibodies—its impact on tumor targeting and strategies for preservation. Cancer Biother Radiopharm 2005;19:669–72.

    Article  Google Scholar 

  28. Leonard JL, Rennke H, Kaplan M, Larsen PR. Subcellular distribution of iodothyronine 5′-deiodinase in cerebral cortex from hypothyroid rats. Biochim Biophys Acta 1982;718:109–19.

    PubMed  CAS  Google Scholar 

  29. Shankar S, Vaidyanathan G, Affleck D, Zalutsky MR. N-Succinimydyl 3-[131I]iodo-4-phosphonomethylbenzoate [131I]SIPMB, a negatively charged substituent-bearing acylation agent for the radioiodination of peptides and mAbs. Bioconjug Chem 2003;14:331–41.

    Article  PubMed  CAS  Google Scholar 

  30. Sheshberadaran H, Payne LG. Protein antigen-monoclonal antibody contact sites investigated by limited proteolysis of monoclonal antibody-bound antigen: protein “footprinting”. Proc Natl Acad Sci USA 1988;85:1–5.

    Article  PubMed  CAS  Google Scholar 

  31. Reilly RM, Sandhu J, Alvarez-Diez T, Gallinger S, Kirsh J, Stern HS. Problems of delivery of monoclonal antibodies-Pharmacokinetic and pharmaceutical solutions. Clin Pharmacokin 1995;28:126–42.

    Article  CAS  Google Scholar 

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Acknowledgements

This project was supported by grants from the Cancer Research Society Inc., the Breast Cancer Society of Canada, and the Ontario Cancer Research Network to R.M.R. and a Predoctoral Traineeship Award from the U.S. Army Breast Cancer Research Program (DAMD 17-02-1-0598) to M.H.

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Authors and Affiliations

  1. Division of Nuclear Medicine, University Health Network, Toronto, ON, Canada

    Meiduo Hu, Paul Chen, Judy Wang, Deborah A. Scollard & Raymond M. Reilly

  2. Department of Radiation Oncology, University Health Network, Toronto, ON, Canada

    Katherine A. Vallis

  3. Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada

    Katherine A. Vallis

  4. Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada

    Meiduo Hu & Raymond M. Reilly

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

    Raymond M. Reilly

  6. Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, ON, Canada, M5S 3M2

    Raymond M. Reilly

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  1. Meiduo Hu
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  2. Paul Chen
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  3. Judy Wang
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  5. Katherine A. Vallis
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Correspondence to Raymond M. Reilly.

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Hu, M., Chen, P., Wang, J. et al. 123I-labeled HIV-1 tat peptide radioimmunoconjugates are imported into the nucleus of human breast cancer cells and functionally interact in vitro and in vivo with the cyclin-dependent kinase inhibitor, p21WAF-1/Cip-1 . Eur J Nucl Med Mol Imaging 34, 368–377 (2007). https://doi.org/10.1007/s00259-006-0189-0

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  • DOI: https://doi.org/10.1007/s00259-006-0189-0

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