SpringerProtocols: Abstract: 1. Functional Analysis of the Antimitogenic Activity of Tumor Suppressors

1. Functional Analysis of the Antimitogenic Activity of Tumor Suppressors

By: Erik S. Knudsen², Steven P. Angus²

Abstract

Full Text

Download PDF (207K)

Loss of tumor suppressors contributes to numerous cancer types. Many, but not all, proteins encoded by tumor suppressor genes have antiproliferative activity and halt cell-cycle progression. In this chapter, we present three methods that have been utilized to monitor the antimitogenic action exerted by tumor suppressors. Tumor suppressor function can be demonstrated by colony formation assays and acquisition of the flat-cell phenotype. Because of the antiproliferative action of these agents, we also present two transient assays that monitor the effect of tumor suppressors on cell-cycle progression. One is based on BrdU incorporation (i.e., DNA replication) and the other on flow cytometry. Together, this triad of techniques is sufficient to determine the action of tumor suppressors and other antiproliferative agents.

Affiliation(s): (2) Department of Cell Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, Cincinnati, OH

Book Title: Cancer Cell Signaling: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 218 | Pub. Date: Dec-09-2002 | Page Range: 3-15 | DOI: 10.1385/1-59259-356-9:03

Subject: Cancer Research

Key Words: Tumor suppressor - green fluorescent protein - bromo-deoxy-uridine - retinoblastoma - cell cycle - cyclin - flow cytometry - mitogen - fluorescence microscopy

References

Download References

1.	Knudson, A. G. (1993) Antioncogenes and human cancer. Proc. Natl. Acad. Sci. USA 90, 10,914–10,921.

2.	Weinberg, R. A. (1995) The retinoblastoma protein and cell cycle control. Cell 81, 323–30.

3.	Macleod, K. (2000) Tumor suppressor genes. Curr. Opin. Genet. Dev. 10, 81–93.

4.	Cavenee, W. K., Dryja, T. P., Phillips, R. A., Benedict, W. F., Godbout, R., Gallie, B. L., et al. (1983) Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature 305, 779–784.

5.	Friend, S. H., Bernards, R., Rogelj, S., Weinberg, R. A., Rapaport, J. M., Albert, D. M., and Dryja, T. P. (1986) A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature 323, 643–646.

6.	Lee, W. H., Bookstein, R., Hong, F., Young, L. J., Shew, J. Y., and Lee, E. Y. (1987) Human retinoblastoma susceptibility gene: cloning, identification, and sequence. Science 235, 1394–1329.

7.	Sherr, C. J. (1996) Cancer cell cycles. Science 274, 1672–1677.

8.	Hanahan, D. and Weinberg, R. A. (2000) The hallmarks of cancer. Cell 100, 57–70.

9.	Evan, G. I. and Vousden, K. H. (2001) Proliferation, cell cycle and apoptosis in cancer. Nature 411, 342–348.

10.	Peltomaki, P. (2001) Deficient DNA mismatch repair: a common etiologic factor for colon cancer. Hum. Mol. Genet. 10, 735–740.

11.	Kolodner, R. D. (1995) Mismatch repair: mechanisms and relationship to cancer susceptibility. Trends Biochem. Sci. 20, 397–401.

12.	Kinzler, K. W. and Vogelstein, B. (1996) Lessons from hereditary colorectal cancer. Cell 87, 159–170.

13.	Levine, A. J. (1997) p53, the cellular gatekeeper for growth and division. Cell 88, 323–331.

14.	Wang, J. Y., Knudsen, E. S., and Welch, P. J. (1994) The retinoblastoma tumor suppressor protein. Adv. Cancer Res. 64, 25–85.

15.	Arap, W., Knudsen, E., Sewell, D. A., Sidransky, D., Wang, J. Y., Huang, H. J., and Cavenee, W. K. (1997) Functional analysis of wild-type and malignant glioma derived CDKN2Abeta alleles: evidence for an RB-independent growth suppressive pathway. Oncogene 15, 2013–2020.

16.	Arap, W., Knudsen, E. S., Wang, J. Y., Cavenee, W. K., and Huang, H. J. (1997) Point mutations can inactivate in vitro and in vivo activities of p16(INK4a)/CDKN2A in human glioma. Oncogene 14, 603–609.

17.	Hinds, P. W., Mittnacht, S., Dulic, V., Arnold, A., Reed, S. I., and Weinberg, R. A. (1992) Regulation of retinoblastoma protein functions by ectopic expression of human cyclins. Cell 70, 993–1006.

18.	Templeton, D. J., Park, S. H., Lanier, L., and Weinberg, R. A. (1991) Nonfunctional mutants of the retinoblastoma protein are characterized by defects in phosphorylation, viral oncoprotein association, and nuclear tethering. Proc. Natl. Acad. Sci. USA 88, 3033–3037.

19.	Knudsen, K. E., Weber, E., Arden, K. C., Cavenee, W. K., Feramisco, J. R., and Knudsen, E. S. (1999) The retinoblastoma tumor suppressor inhibits cellular proliferation through two distinct mechanisms: inhibition of cell cycle progression and induction of cell death. Oncogene 18, 5239–45.

20.	Qin, X. Q., Chittenden, T., Livingston, D. M., and Kaelin, W. G. Jr. (1992) Identification of a growth suppression domain within the retinoblastoma gene product. Genes Dev. 6, 953–964.

21.	Knudsen, E. S., Pazzagli, C., Born, T. L., Bertolaet, B. L., Knudsen, K. E., Arden, K. C., et al. (1998) Elevated cyclins and cyclin-dependent kinase activity in the rhabdomyosarcoma cell line RD. Cancer Res. 58, 2042–2049.

22.	Knudsen, E. S., Buckmaster, C., Chen, T. T., Feramisco, J. R., and Wang, J. Y. (1998) Inhibition of DNA synthesis by RB: effects on G1/S transition and S-phase progression. Genes Dev. 12, 2278–2292.

23.	Knudsen, K. E., Fribourg, A. F., Strobeck, M. W., Blanchard, J. M., and Knudsen, E. S. (1999) Cyclin A is a functional target of retinoblastoma tumor suppressor protein-mediated cell cycle arrest. J. Biol. Chem. 274, 27,632–27,641.

24.	Agami, R. and Bernards, R. (2000) Distinct initiation and maintenance mechanisms cooperate to induce G1 cell cycle arrest in response to DNA damage. Cell 102, 55–66.

25.	Zhu, L., van den Heuvel, S., Helin, K., Fattaey, A., Ewen, M., Livingston, D., et al. (1993) Inhibition of cell proliferation by p107, a relative of the retinoblastoma protein. Genes Dev. 7, 1111–1125.

26.	Kanda, T., Sullivan, K. F., and Wahl, G. M. (1998) Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells. Curr. Biol. 8, 377–385.

27.	Morgenstern, J. P. and Land, H. (1990) Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 18, 3587–3596.

Comments (Loading...)

Post comment/View all comments