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  • Acquired Diseases
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Anti-tumorigenic effect of a K-ras ribozyme against human lung cancer cell line heterotransplants in nude mice

Gene Therapy volume 7, pages 2041–2050 (2000)Cite this article

Abstract

Approximately 15–30% of human non-small cell lung cancers (NSCLC) carry K-ras mutations, among which point mutations at codon 12 are the most common. This study characterizes the anti-tumor effect of an anti-K-ras ribozyme adenoviral vector (KRbz-ADV; replication-deficient, E1-deleted Ad5 backbone) against NSCLC lines that express the relevant mutation (K-ras codon 12 GGT → GTT; H441 and H1725). KRbz-ADV significantly inhibited tumor cell growth (38–94% reduction by 3H-thymidine uptake) in a time- and dose-dependent manner, but produced minimal growth inhibition on normal epithelial cells, or NSCLC H1650 cells that lack the relevant mutation. The in vivo anti-tumorigenic effect of KRbz-ADV treatment was characterized with cell line xenografts in nu/nu mice. Pre-treatment with KRbz-ADV (10 or 20 p.f.u. per cell) completely abrogated subcutaneous engraftment of H441 (n = 13) or H1725 cells (n = 8), as compared with a 100% tumor take and progressive tumor growth in animals that received untreated tumor cells, or control vector (luciferase-adenovirus/Luc-ADV)-treated tumor cells. Pre-treatment with a mutant anti-K-ras ribozyme adenoviral vector (mutKRbz-ADV), which has the same specificity as KRbz but lacks ribozyme catalytic activity, did not produce an anti-tumorigenic effect. The in vivo effect of KRbz-ADV treatment was further examined by initiating injections (2 × 109 p.f.u.) at 7 days after tumor induction. Pre-existing tumor growth was reduced by 39% by a single intratumoral injection. Repeat injections (three or five KRbz-ADV-intratumoral injections at 2 × 109 p.f.u. every other day) resulted in complete tumor regression in five of seven mice. In contrast, single or multiple injections of control vector Luc-ADV did not significantly alter tumor xenograft outcome. Ribozyme expression was confirmed in H441 cells that demonstrated reduced growth after KRbz-ADV treatment. Reduced growth corresponded to significantly lowered levels of K-ras mRNA, as defined by RT-PCR (51% of untreated level, n = 3) and RNase protection assay (56% of untreated level, n = 4) analyses. Further, 37.5% of KRbz-ADV-treated cells underwent apoptosis, as compared with 11.7%, and 19.0% in untreated and Luc-ADV-treated cultures, respectively. A significantly higher proportion of KRbz-ADV-treated H441 cells (58.2%) underwent apoptosis when maintained under anchor-independent conditions that simulate in vivo tumorigenesis (‘anoikis’). This is the first report that demonstrates that KRbz-ADV can effectively inhibit in vivo tumorigenesis, and produces regression of pre-existing human lung tumor xenografts having the relevant K-ras mutation.

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Acknowledgements

This work was supported in part by the Mary Crowley Research Foundation, the Robert A Schanbaum Memorial Fund for Cancer Research, and by a Berlex Biosciences Cancer Research grant. The authors thank Guido A Ordonez of the Immunology Laboratory, Baylor University Medical Center, for his assistance on flow cytometric analysis of apoptosis. We thank Joseph M Lawson and Beverly Peters, Baylor University Medical Center, for their assistance with preparation of the figures.

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Authors and Affiliations

  1. Mary C Crowley Cancer Research Program, Baylor Research Institute, Baylor University Medical Center, Dallas, TX, USA

    Y-A Zhang, J Nemunaitis & AW Tong

  2. US Oncology, Inc., Dallas, TX, USA

    J Nemunaitis

  3. Berlex Biosciences, Richmond, CA, USA

    KJ Scanlon

  4. Cancer Immunology Research Laboratory, Baylor-Sammons Cancer Center, Baylor University Medical Center, Dallas, TX, USA

    AW Tong

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Zhang, YA., Nemunaitis, J., Scanlon, K. et al. Anti-tumorigenic effect of a K-ras ribozyme against human lung cancer cell line heterotransplants in nude mice. Gene Ther 7, 2041–2050 (2000). https://doi.org/10.1038/sj.gt.3301331

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