Colony Growth Suppression by Tumor Suppressor Genes

  • Gen Sheng Wu
Part of the Methods in Molecular Biology™ book series (MIMB, volume 223)

Abstract

The ability of a tumor suppressor gene to inhibit cell growth is critical for its tumor suppression. Such activity is carried out largely by inducing cell cycle arrest and apoptosis. For example, when the p53 tumor suppressor gene is activated by DNA damage (1), it inhibits the growth of tumor cells by inducing cell cycle arrest or apoptosis (2). Despite a temporal and reversible event, cell cycle arrest allows damaged DNA to be repaired and thereby prevents abnormal DNA propagation and the emergence of cancer cells. On the other hand p53-induced apoptosis is powerful and irreversible (3). When p53 is introduced into tumor cells, for example via p53 adenovirus infection, the cells undergo rapidly apoptosis (4). Conversely, loss of p53-mediated apoptosis has been implicated not only in tumor progression, but also in the drug-resistant phenotype of tumor cells (5). Thus, the tumor suppressor gene often possesses these two features. Accordingly, several techniques have been designed to analyze cell cycle arrest and apoptosis induction to characterize the growth inhibition activity of tumor suppressor genes, including flow cytometry assay (6), MTT assay (7), and clonogenic survival assay (8). Among these, the clonogenic survival assay is the most commonly used assay to assess growth inhibition in vitro. Basically, this method examines how the gene of interest affects cell growth in cultured cells. It includes three steps. The first step is to construct a mammalian expression vector that can express the gene of interest. This involves standard molecular cloning techniques. The second step introduces the expression vector into human cancer cell lines via a carrier such as lipofectin.

Keywords

Agar Ampicillin Ligase 

References

  1. 1.
    Kastan, M. B., Onyekwere, O., Sidransky, D., Vogelstein, B., and Craig, R. W. (1991) Participation of p53 protein in the cellular response to DNA damage. Cancer Res. 51, 6304–6311.PubMedGoogle Scholar
  2. 2.
    Levine, A. J. (1997) p53, the cellular gatekeeper for growth and division. Cell 88, 323–331.PubMedCrossRefGoogle Scholar
  3. 3.
    El-Deiry, W. S. (1998) Regulation of p53 downstream genes. Semin. Cancer Biol. 8, 345–357.PubMedCrossRefGoogle Scholar
  4. 4.
    Blagosklonny, M. V and El-Deiry, W. S. (1996) In vitro evaluation of a p53-expressing adenovirus as an anti-cancer drug. Int. J. Cancer 67, 386–392.PubMedCrossRefGoogle Scholar
  5. 5.
    Lowe, S. W. and Lin, A. W. (2000) Apoptosis in cancer. Carcinogenesis 21, 485–495.PubMedCrossRefGoogle Scholar
  6. 6.
    Macey, M. G. (1988) Flow cytometry: principles and clinical applications. Med. Lab. Sci. 45, 165–173.PubMedGoogle Scholar
  7. 7.
    Carmichael, J., DeGraff, W. G., Gazdar, A. F., Minna, J. D., and Mitchell, J. B. (1987) Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 47, 936–942.PubMedGoogle Scholar
  8. 8.
    El-Deiry, W. S., Tokino, T, Velculesue, V E., et al. (1993) WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817–825.PubMedCrossRefGoogle Scholar
  9. 9.
    Sambrook, J., Fritsch, E. F, and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar

Copyright information

© Humana Press Inc. 2003

Authors and Affiliations

  • Gen Sheng Wu
    • 1
  1. 1.Karmanos Cancer Institute, Department of PathologyWayne State University School of MedicineDetroit

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