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Virus-mediated killing of cells that lack p53 activity

Nature volume 412, pages 914–917 (2001)Cite this article

An Addendum to this article was published on 14 March 2002

Abstract

A major goal of molecular oncology is to identify means to kill cells lacking p53 function. Most current cancer therapy is based on damaging cellular DNA by irradiation or chemicals. Recent reports1,2 support the notion that, in the event of DNA damage, the p53 tumour-suppressor protein is able to prevent cell death by sustaining an arrest of the cell cycle at the G2 phase. We report here that adeno-associated virus (AAV) selectively induces apoptosis in cells that lack active p53. Cells with intact p53 activity are not killed but undergo arrest in the G2 phase of the cell cycle. This arrest is characterized by an increase in p53 activity and p21 levels and by the targeted destruction of CDC25C. Neither cell killing nor arrest depends upon AAV-encoded proteins. Rather, AAV DNA, which is single-stranded with hairpin structures at both ends3,4, elicits in cells a DNA damage response that, in the absence of active p53, leads to cell death. AAV inhibits tumour growth in mice. Thus viruses can be used to deliver DNA of unusual structure into cells to trigger a DNA damage response without damaging cellular DNA and to selectively eliminate those cells lacking p53 activity.

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Figure 1: DNA content of cells after AAV infection.
Figure 2: Biochemical analyses of proteins in AAV-infected cells.
Figure 3

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Acknowledgements

We thank N. Paduwat and B. Bentele for technical assistance; M. Chapman and X. Qing for the gift of AAV-2; B. Vogelstein for the HCT116 cell line and its derivatives; R. Iggo for antibodies and the Saos-2 and U2OS cell lines; M. Oren for p53DD vectors; A. M. R. Taylor for the ATM-null cells; K. Alevizopoulos for retroviral vectors; J. Kleinschmidt for recombinant baculovirus; C. Wenz for assistance in insect cell culture; and S. Gasser, A.-L. Ducrest, M. Höss, B. Hirt, J. Jiricny, R. Iggo and V. Simanis for discussions. B. Sordat and F. Hoffmann helped with the animal experiments, and P. Reichenbach and J. Wyniger with the micro-injection. This work was supported by Cancer Research Switzerland and the Swiss National Science Foundation.

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  1. Swiss Institute for Experimental Cancer Research (ISREC), 155, Ch. des Boveresses, Epalinges, 1066, Switzerland

    Kenneth Raj, Phyllis Ogston & Peter Beard

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  1. Kenneth Raj
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  2. Phyllis Ogston
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Corresponding author

Correspondence to Peter Beard.

Supplementary information

Figure 1

(JPG 33.2 KB)

Effects of AAV-2 infection on osteosarcoma cells Schematic representation of AAV DNA. (a) Saos-2 cells (b-d) or U2OS cells (e-g) are shown either uninfected or after infection with AAV at a mul tiplicity of i fection (MOI) 5000. All photographs are at the same magnification.

Figure 2a

Effect of AAV on tumour formation (JPG 32.2 KB)

HCT116p53-/-or HCT116p53+/+cells (one million cells in 50 µl DMEM) were injected under the skin of both flanks of nude mice. Two days later the right flanks were injected with AAV (5X10 8 virus particles) while the left flanks were injected with PBS. Approximately 20 days later, the mice were examined for tumours. Examples of mice with HCT116p53-/-tumours (1 and 2)and HCT116p53+/+ tumours (3 and 4) are shown.

Figure 2b

Effect of AAV on established tumours (JPG 25.1 KB)

Tumours were induced in both flanks of nude mice by injecting either HCT116p53-/-ells or another p53-negative colon carcinoma cell line, HT29 (one million cells in 50 µl DMEM). After approximately 10 to 14 days, the tumours on the right flanks were injected with AAV (5x10 9virus particles) while tumours on the left flanks were injected with PBS on alternate days for 12 days. Examples of results with HT29 are shown. Mouse (1) is a control where tumours on both flanks were not injected. Mice (2-5) were injected as describe above. Tumours injected with AAV in mice 2 and 3 regressed completely while those of mice 4 and 5 grew more slowly in comparison to tumours injected with PBS.

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Raj, K., Ogston, P. & Beard, P. Virus-mediated killing of cells that lack p53 activity. Nature 412, 914–917 (2001). https://doi.org/10.1038/35091082

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