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A novel mammalian cell-based approach for the discovery of anticancer drugs with reduced cytotoxicity on non-dividing cells

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Abstract

A key asset of cytotoxic drugs in cancer therapeutics is their ability to discriminate between proliferating and mitotically inert cells and eliminate preferentially neoplastic ones. We have designed a high throughput-compatible mammalian cell-based assay for the discovery of cytotoxic drugs, which selectively kill proliferation-competent target cells. This cytotoxic drug discovery assay is based on a transgenic CHO-K1-derived cell line engineered for a conditional G1-specific growth arrest following tetracycline-responsive overexpression of the human cyclin-dependent kinase inhibitor p27Kip1. The CHO-derived cell line CHO-p27Kip1 shows wild type proliferation rates and can be expanded in the presence of tetracycline antibiotics when p27Kip1 expression is repressed. Upon withdrawal of regulating antibiotics CHO-p27Kip1 differentiates into a 1:1 mixed population consisting of two different proliferation phenotypes: (i) a G1-arrested cell population induced by heterologous expression of p27Kip1 which mimics mitotically inactive terminally differentiated cells and (ii) a proliferation-competent cell population which eliminated the p27Kip1 expression unit and imitates neoplastic cell characteristics. Addition of chemical or metabolic libraries to CHO-p27Kip1 populations cultivated in tetracycline-free medium followed by scoring for cell viability will reveal cytotoxic drug candidates associated with a high viability ratio of proliferation-competent/arrested populations.

We have validated the cell-based cytotoxic drug discovery assay using the clinically licensed cancer drugs mitomycin C, doxorubicin, etoposide and 5-fluorouracil. Comparative proof-of-concept studies showed that these top-prescribed cancer therapeutics preferentially eliminate proliferating cells while showing less interference with the viability of G1-arrested cell populations. These results demonstrate the CHO-p27Kip1-based cytotoxic drug finder technology is ready-to-apply for high throughput screenings of chemical as well as metabolic libraries to discover novel cancer therapeutics which show reduced cytotoxicity on terminally differentiated cells.

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References

  1. Balmain A, Gray J, Ponder B: The genetics and genomics of cancer. Nature Genet 33: 238–244, 2003

    Article  CAS  PubMed  Google Scholar 

  2. Ohlsson R, Kanduri C, Whitehead J, Pfeifer S, Lobanenkov V, Feinberg AP: Epigenetic variability and the evolution of human cancer. Adv Cancer Res 88: 145–168, 2003

    CAS  PubMed  Google Scholar 

  3. Neiman PE, Gribic JJ, Polony TS, Kimmel R, Bowers SJ, Delrow J, Beemon KL: Functional genomic analysis reveals distinct neoplastic phenotypes associated with c-myb mutation in the bursa of Fabricius. Oncogene 22: 1073–1086, 2003

    Article  CAS  PubMed  Google Scholar 

  4. Vincent-Salomon A, Thiery JP: Host microenvironment in breast cancer development: Epithelial-mesenchymal transition in breast cancer development. Breast Cancer Res 5: 101–106, 2003

    Article  CAS  PubMed  Google Scholar 

  5. Jaenisch R, Bird A: Epigenetic regulation of gene expression: How the genome integrates intrinsic and environmental signals. Nat Genet 33: 245–254, 2003

    Article  CAS  PubMed  Google Scholar 

  6. Garsky VM, Lumma PK, Feng DM, Wai J, Ramjit HG, Sardana MK, Oliff A, Jones RE, DeFeo-Jones D, Freidinger RM: The synthesis of a prodrug of doxorubicin designed to provide reduced systemic toxicity and greater target efficacy. J Med Chem 44: 4216–4224, 2001

    Article  CAS  PubMed  Google Scholar 

  7. Hosie KB, Kerr DJ, Gilbert JA, Downes M, Lakin G, Pemberton G, Timms K, Young A, Stanley A: A pilot study of adjuvant intraperitoneal 5-fluorouracil using 4% icodextrin as a novel carrier solution. Eur J Surg Oncol 29: 254–260, 2003

    Article  CAS  PubMed  Google Scholar 

  8. Canellos GP, Gollub J, Neuberg D, Mauch P, Shulman LN: Primary systemic treatment of advanced Hodgkin’s disease with EVA (etoposide, vinblastine, doxorubicin): 10-year follow-up. Ann Oncol 14: 268–272, 2003

    Article  CAS  PubMed  Google Scholar 

  9. Chin L, Tam A, Pomerantz J, Wong M, Holash J, Bardeesy N, Shen Q, O’Hagan R, Pantginis J, Zhou H, Horner JW, Cordon-Cardo C, Yancopoulos GD, DePinho RA: Essential role for oncogenic Ras in tumor maintenance. Nature 400: 468–472, 1999

    Article  CAS  PubMed  Google Scholar 

  10. Fussenegger M, Schlatter S, Datwyler D, Mazur X, Bailey JE: Controlled proliferation by multigene metabolic engineering enhances the productivity of Chinese hamster ovary cells. Nat Biotecnol 16: 468–472, 1998

    Article  CAS  Google Scholar 

  11. Umaña P, Jean-Mairet J, Moudry R, Amstutz H, Bailey JE: Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cytotoxic activity. Nat Biotechnol 17: 176–180, 1999

    Article  PubMed  Google Scholar 

  12. Aubel D, Morris R, Lennon B, Rimann M, Kaufmann H, Folcher M, Bailey JE, Thompson CJ, Fussenegger M: Design of a novel mammalian screening system for the detection of bioavailable non-cytotoxic streptogramin antibiotics. J Antibiot 54: 44–55, 2001

    CAS  PubMed  Google Scholar 

  13. Fussenegger M: The impact of mammalian gene regulation concepts on functional genomic research, metabolic engineering, and advanced gene therapies. Biotechnol Prog 17: 1–51, 2001

    CAS  PubMed  Google Scholar 

  14. Qian F, Pan W: Construction of a tetR-integrated Salmonella enterica serovar Typhi CVD908 strain that tightly controls expression of the major merozoite surface protein of Plasmodium falciparum for applications in human Vaccine production. Infect Immun 70: 2029–2038, 2002

    CAS  PubMed  Google Scholar 

  15. Ozawa CR, Springer ML, Blau HM: A novel means of drug delivery: Myoblast-mediated gene therapy and regulatable retroviral vectors. Annu Rev Pharmacol Toxicol 40: 295–317, 2000

    CAS  PubMed  Google Scholar 

  16. Hillen W, Klock G, Kaffenberger I, Wray LV, Reznikoff WS: Purification of the TET repressor and TET operator from the transposon Tn10 and characterization of their interaction. J Biol Chem 257: 6605–6613, 1982

    CAS  PubMed  Google Scholar 

  17. Gossen M, Bujard H: Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89: 5547–5551, 1992

    CAS  PubMed  Google Scholar 

  18. Martinez LA, Chen Y, Fischer S, Conti C: Coordinated changes in cell cycle machinery occur during keratinocyte terminal differentiation. Oncogene 18: 397–406, 1999

    CAS  PubMed  Google Scholar 

  19. Chellapan SP, Giordano A, Fischer PB: Role of cyclin-dependent kinases and their inhibitors in cellular differentiation and development. Curr Top Micro Immunol 227: 57–103, 1998

    Google Scholar 

  20. Vozza A, Borriello A, Criniti V, Vozza G, Della-Ragione F: New established melanoma cell lines: Genetic and biochemical characterization of cell division cycle. J Eur Acad Dermatol Venereol 17: 37–41, 2003

    CAS  PubMed  Google Scholar 

  21. Yang C, Sun M, Ilic Z, Friedrich TD, Sell S: Reduced expression of p27kip1 and increased hepatocyte proliferation in p53-deficient mice. Mol Carcinog 36: 15–22, 2003

    CAS  PubMed  Google Scholar 

  22. Cho NH, Kim YT, Kim JW: Alteration of cell cycle in cervical tumor associated with human papillomavirus: Cyclin-dependent kinase inhibitors. Yonsei Med J 43: 722–728, 2003

    Google Scholar 

  23. Kato JY, Matsuoka M, Polyak K, Massagué J, Sherr CJ: Cyclin AMP-induced G1 phase arrest mediated by an inhibitor (p27Kip1) of cyclin-dependent kinase 4 activation. Cell 79: 487–496, 1994

    CAS  PubMed  Google Scholar 

  24. Polyak K, Lee ML, Erdjument-Bromage H, Koff A, Roberts JM, Tempst P, Massagué J: Cloning of p27Kip1, a cyclin-dependent kinase I inhibitor and a potential mediator of extracellular antimitogenic signals. Cell 78: 59–66, 1994

    CAS  PubMed  Google Scholar 

  25. Ladha MH, Lee KY, Upton TM, Reed MF, Ewen ME: Regulation of exit from quiescence by p27 and cyclin D1-CDK4. Mol Cell Biol 18: 6605–6615, 1998

    CAS  PubMed  Google Scholar 

  26. Lowenheim H, Furness DN, Kil J, Zinn C, Gultig K, Fero ML, Frost D, Gummer AW, Roberts JM, Rubel EW, Hackney CM, Zenner HP: Gene disruption of p27(Kip1) allows cell proliferation in the postnatal and adult organ of corti. Proc Natl Acad Sci USA 96: 4084–4088, 1999

    CAS  PubMed  Google Scholar 

  27. Rivard N, Boucher MJ, Asselin C, L’Allemain G: MAP kinase cascade is required for p27 downregulation and S phase entry in fibroblasts and epithelial cells. Am J Physiol 277: 652–664, 1999

    Google Scholar 

  28. Park HK, Lee KW, Choi JS, Joo CK: Mitomycin C-induced cell death in mouse lens epithelial cells. Ophthalmic Res 34: 213–219, 2002

    CAS  PubMed  Google Scholar 

  29. Corbett T, Valeriote F, LoRusso P, Polin L, Panchapor C, Pugh S, White K, Knight J, Demchik L, Jones J, Jones L, Lisow L (eds): In vivo methods for screening and preclinical testing. Drug Development Guide. Humana Press Inc, Totowa, NJ, 1997, pp 75–99

  30. Steff AM, Fortin M, Arguin C, Hugo P: Detection of a decrease in green fluorescent protein fluorescence for the monitoring of cell death: An assay amenable to high-throughput screening technologies. Cytometry 45: 237–243, 2001

    CAS  PubMed  Google Scholar 

  31. Sohn TA, Su GH, Ryu B, Yeo CJ, Kern SE: High-throughput drugscreening of the DPC4 tumor-suppressor pathway in human pancreatic cancer cells. Ann Surg 233: 696–703, 2001

    CAS  PubMed  Google Scholar 

  32. Mosmann T: Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 16: 55–63, 1983

    Google Scholar 

  33. Scudiero DA, Shoemaker RH, Paul KD, Monks A, Tierney S, Nofziger TH, Currens MJ, Seniff D, Boyd MR: Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res 48: 4827–4833, 1988

    CAS  PubMed  Google Scholar 

  34. Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR: New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 82: 1107–1112, 1990

    CAS  PubMed  Google Scholar 

  35. Allison DC, Ridolpho P: Use of a trypan blue assay to measure the deoxyribonucleic acid content and radioactive labeling of viable cells. J Histochem Cytochem 28: 700–703, 1980

    CAS  PubMed  Google Scholar 

  36. Reynolds CP, Black AT, Woody JN: Sensitive method for detecting viable cells seeded into bone marrow. Cancer Res 46: 5878–5881, 1986

    CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Hoenggerberg, HPT D74, CH-8093, Zurich, Switzerland

    Valeria Gonzalez-Nicolini, Cornelia Fux & Martin Fussenegger

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  1. Valeria Gonzalez-Nicolini
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  2. Cornelia Fux
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  3. Martin Fussenegger
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Gonzalez-Nicolini, V., Fux, C. & Fussenegger, M. A novel mammalian cell-based approach for the discovery of anticancer drugs with reduced cytotoxicity on non-dividing cells. Invest New Drugs 22, 253–262 (2004). https://doi.org/10.1023/B:DRUG.0000026251.00854.77

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