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Mutational Analysis of the Androgen Receptor Using Laser Capture Microdissection and Direct Sequencing

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Cancer Cell Signaling

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 218))

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Abstract

Molecular analysis of prostate cancer specimens is complicated by tumor heterogeneity and admixture of malignant cells with benign epithelium, stroma and inflammatory cells. Laser Capture Microdissection allows procurement of selected groups of cells from specific areas of tissue sections. We have used Laser Capture Microdissection to obtain pure samples of malignant prostate epithelial cells from frozen radical prostatectomy specimens. Methods for DNA extraction, androgen receptor amplification and exon sequencing have been optimized. These methods should be adaptable for molecular analysis of DNA, RNA and protein from other complex tissues.

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References

  1. Jemal, A., Thomas, A., Murray, T., and Thun, M. (2002) Cancer statistics. Ca. Cancer J. Clin. 52, 23–47.

    Article  PubMed  Google Scholar 

  2. Feldman, B. J. and Feldman, D. (2001) The development of androgen-independent prostate cancer. Nature Rev. 1, 34–35.

    Article  CAS  Google Scholar 

  3. Veldscholte, J., Ris-stapler, C., Kuiper, G. G. J. M., Jenster, G., Berrevoets, C. A., Claassen, E., et al. (1990) A mutation in the ligand binding domain of the androgen receptor of human LNCaP cells affects steroid binding characteristics and response to anti-androgens. Biochem. Biophys. Res. Commun. 173, 534–540.

    Article  PubMed  CAS  Google Scholar 

  4. Harris, S. E., Harris, M. A., Rong, Z. X., Hall, J. A., Judge, S., French, F. S., et al. (1991) Androgen regulation of HBGF-1(aFGF) mRNA and characterization of the androgen receptor mRNA in the human prostate carcinoma cell line LNCaP/A-DEP, in Molecular and Cellular Biology of Prostate Cancer (Karr, J. P., Coffey, D. S., and Smith, R. G., eds.), Plenum, New York, pp. 315–330.

    Google Scholar 

  5. Tilley, W. D., Buchanan, G., Hickey, T. E., and Bentel, J. M. (1996) Mutations in the androgen receptor gene are associated with progression of human prostate cancer to androgen independence. Clin. Cancer Res. 2, 277–285.

    PubMed  CAS  Google Scholar 

  6. Takahashi, H., Furusato, M., Allsbrook, W. C. Jr., Nishii, H., Wakui, S., Barrett, J. C., and Boyd, J. (1995) Prevalence of androgen receptor gene mutations in latent prostatic carcinomas from Japanese men. Cancer Res. 55, 1621–1624.

    PubMed  CAS  Google Scholar 

  7. Suzuki, H., Sato, N., Watabe, Y., Masai, M., Seino, S., and Shimazaki, J. (1993) Androgen receptor gene mutations in human prostate cancer. J. Steroid Biochem. Mol. Biol. 46, 759–765.

    Article  PubMed  CAS  Google Scholar 

  8. Elo, J. P., Kvist, L., Leinonen, K., Isomaa, V., Henttu, P., Lukkarinen, O., and Vehko, P. (1995) Mutated human androgen receptor gene detected in a prostatic cancer patient is also activated by estradiol. J. Clin. Endocrinol. Metab. 80, 3494–3500.

    Article  PubMed  CAS  Google Scholar 

  9. Newmark, J. R., Hardy, D. O., Tonb, D. C., Carter, B. S., Epstein, J., Isaacs, W. B., et al. (1992) Androgen receptor gene mutations in human prostate cancer. Proc. Natl. Acad. Sci. USA 89, 6319–6323.

    Article  PubMed  CAS  Google Scholar 

  10. Culig, Z., Hobisch, A., Cronauer, M. V., Cato, A. C. B., Hittmair, A., Radmayr, C., et al. (1993) Mutant androgen receptor detected in an advanced-stage prostatic carcinoma is activated by adrenal androgens and progesterone. Mol. Endocrinol. 8808–8809, 1541–1550.

    Article  Google Scholar 

  11. Taplin, M. E., Bubley, G. J., Shuster, T. D., Frantz, M. E., Spooner, A. E., Ogata, G. K., et al. (1995) Mutation of the androgen-receptor gene in metastatic androgen-independent prostate cancer. N. Engl. J. Med. 332, 1393–1398.

    Article  PubMed  CAS  Google Scholar 

  12. Kleinerman, D. I., Troncoso, P., Pisters, L. L., Sleddens, H. M. E. B., Navone, N. M., Ordonez, van der Kwast, T. H., et al. (1994) Evaluation of androgen receptor expression and structure in bone metastases of prostate carcinoma. Basic and clinical aspects of prostate cancer: An AACR Special Conference, Rancho Mirage CA, Dec. 8–12 (A-22).

    Google Scholar 

  13. Ruizeveld de Winter, J. A., Janssen, P. J., Sleddens, H. M., Verleun-Mooijman, M. C., Trapman, J., Brinkmann, A. O., et al. (1994) Androgen receptor status in localized and locally progressive hormone refractory human prostate cancer. Am. J. Pathol. 144, 735–746.

    PubMed  CAS  Google Scholar 

  14. Gaddipati, J. P., McLeod, D. G., Heidenberg, H. B., Sesterhenn, M., Finger, M. J., Moul, J. W., and Srivastava, S. (1994) Frequent detection of codon 877 mutation in the androgen receptor gene in advanced prostate cancers. Cancer Res. 54, 2861–2864.

    PubMed  CAS  Google Scholar 

  15. Evans, B. A. J., Harper, M. E., Daniells, C. E., Watts, C. E., Matenhelia, S., Green, J., and Griffiths, K. (1996) Low incidence of androgen receptor gene mutations in human prostatic tumors using single strand conformation polymorphism analysis. Prostate 28, 162–171.

    Article  PubMed  CAS  Google Scholar 

  16. Kleinerman, D. I., Troncoso, P., Pisters, L. L., et al. (1996) Expression and structure of the androgen receptor in bone metastases of hormone refractory prostate cancer. J. Urol. 155, 626A.

    Google Scholar 

  17. Peterziel, H., Culig, Z., Stober, J., Hobisch, A., Radmayr, C., Bartsch, G., et al. (1995) Mutant androgen receptors in prostatic tumors distinguish between aminoacid-sequence requirements for transactivation and ligand binding. Int. J. Cancer 63, 544–550.

    Article  PubMed  CAS  Google Scholar 

  18. Tan, J.-A., Sharief, Y., Hamil, K. G., Gregory, C. W., Zang, D.-Y., Sar, M., et al. (1997) Dehydroepiandrosterone activates mutant androgen receptors expressed in the androgen dependent human prostate cancer xenograft CWR22 and LNCaP cells. Mol. Endocrinol. 11, 450–459.

    Article  PubMed  CAS  Google Scholar 

  19. Emmert-Buck, M. R., Bonner, R. F., Smith, P. D., Chaquil, R. F., Zhuang, Z., Goldstein, S. R., et al. (1996) Laser Capture Microdissection. Science 274, 998–1001.

    Article  PubMed  CAS  Google Scholar 

  20. Rubin, M. A. (2001) Use of laser capture microdissection, cDNA microarrays, and tissue microarrays in advancing our understanding of prostate cancer. J. Pathol. 195, 80–86.

    Article  PubMed  CAS  Google Scholar 

  21. Goldstein, S. R., McQueen, P. G., and Bonner, R. F. (1998) Thermal modeling of Laser Capture Microdissection. Appl. Opt. 37, 7378–7391.

    Article  PubMed  CAS  Google Scholar 

  22. Lubahn, D. B., Joseph, D. R., Sar, M., Tan, J.-A., Higgs, H. N., Larson, R. E., et al. (1988) The human androgen receptor: complementary deoxyribonucleic acid cloning, sequence analysis and gene expression in prostate. Mol. Endocrinol. 2, 1265–1275.

    Article  PubMed  CAS  Google Scholar 

  23. Williams, C., Ponten, F., Moberg, C., Soderkvist, P., Uhlen, M., Ponten, J., et al. (1999) A high frequency of sequence alterations is due to formalin fixation of archival specimens. Am. J. Pathol. 155, 1467–1471.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Division of Urology, Department of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, NC

    Sheila Greene, Olga G. Kozyreva, Kris E. Gaston & James L. Mohler

  2. Laboratory of Experimental Pathology, National Institute of Environmental Health Sciences, Research Triangle Park, NC

    Patricia Stockton & Gordon P. Flake

  3. UNC-Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC

    Kris E. Gaston & James L. Mohler

Authors
  1. Sheila Greene
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  2. Patricia Stockton
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  3. Olga G. Kozyreva
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  4. Kris E. Gaston
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  5. Gordon P. Flake
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  6. James L. Mohler
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Editor information

Editors and Affiliations

  1. Brody School of Medicine, East Carolina University, Greenville, NC

    David M. Terrian

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© 2003 Humana Press Inc., Totowa, NJ

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Greene, S., Stockton, P., Kozyreva, O.G., Gaston, K.E., Flake, G.P., Mohler, J.L. (2003). Mutational Analysis of the Androgen Receptor Using Laser Capture Microdissection and Direct Sequencing. In: Terrian, D.M. (eds) Cancer Cell Signaling. Methods in Molecular Biology™, vol 218. Humana Press. https://doi.org/10.1385/1-59259-356-9:287

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  • DOI: https://doi.org/10.1385/1-59259-356-9:287

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-075-5

  • Online ISBN: 978-1-59259-356-9

  • eBook Packages: Springer Protocols

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