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A sensitive dual-fluorescence reporter system enables positive selection of ras suppressors by suppression of ras-induced apoptosis

Cancer Gene Therapy volume 10, pages 745–754 (2003)Cite this article

Abstract

We have developed a novel dual-fluorescence reporter system incorporating green (GFP) and red (RFP) fluorescent proteins to monitor expression of the N-rasm gene and an N-rasm suppressor, respectively. Retroviral vectors were produced in which human N-rasm (codon 13 mutation) was coexpressed with GFP, and a ribozyme specifically targeting N-rasm was coexpressed with RFP. N-Rasm suppression was monitored by measurement of GFP fluorescence in dual-fluorescent (GFP and RFP) cells. We demonstrated that the degree of N-rasm suppression was dependent on the ribozyme dose, proportional to red fluorescence, in dual-fluorescent cells. We further showed that ribozyme-mediated N-rasmsuppression inhibited growth of NIH3T3 and CD34-positive TF-1 cells. In these cultures, ras suppressor activity resulted in the depletion of suppressor-positive cells due to inhibition of cell growth. In contrast, N-rasm suppression produced a growth advantage to human leukemic K562 cells, presumably by inhibiting N-rasm-induced apoptosis. In K562 cells, ras suppression resulted in the outgrowth of suppressor-positive cells. This provides a platform to identify suppressors of ras that is based on function.

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Acknowledgements

We thank Dr M Grez for the generous supply of N-rasm encoding plasmid and Johnson & Johnson Research for facility space and staff discussions. GS is a Principal Research Fellow of the NHMRC of Australia and this work was funded by a project grant of the NHMRC.

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Authors and Affiliations

  1. School of Physiology and Pharmacology, The University of New South Wales, Sydney, NSW, Australia

    Alla Dolnikov, Sylvie Shen, Michelle Millington, Toby Passioura & Geoff Symonds

  2. Department of Clinical Pharmacology and Toxicology, St Vincent's Hospital, Darlinghurst, NSW, Australia

    Alla Dolnikov, Sylvie Shen, Michelle Millington & Geoff Symonds

  3. Children's Cancer Institute Australia, Randwick, Sydney, 2031, NSW, Australia

    Alla Dolnikov, Sylvie Shen, Michelle Millington, Toby Passioura & Geoff Symonds

  4. The Centenary Institute of Cancer Medicine and Cell Biology, University of Sydney, Camperdown, Australia

    Michelle Pedler & John Edward Joshua Rasko

  5. Sydney Cancer Centre, Royal Prince Alfred Hospital, Camperdown, Australia

    Michelle Pedler & John Edward Joshua Rasko

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  1. Alla Dolnikov
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  2. Sylvie Shen
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Correspondence to Geoff Symonds.

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Dolnikov, A., Shen, S., Millington, M. et al. A sensitive dual-fluorescence reporter system enables positive selection of ras suppressors by suppression of ras-induced apoptosis. Cancer Gene Ther 10, 745–754 (2003). https://doi.org/10.1038/sj.cgt.7700603

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